Agrobacterium for transient transfection of whole plants

ABSTRACT

A process of transiently transfecting a plant or leaves on a plant, comprising contacting said plant or said leaves with a suspension comprising  Agrobacterium  cells of strain CryX or a derivative strain of strain CryX, wherein said derivative strain has the chromosomal background of strain CryX or said derivative strain contains the vir plasmid of strain CryX or a derivative of said vir plasmid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/386,441, which was filed on Sep. 19, 2014, has a § 371(c) date ofSep. 19, 2014, and is the U.S. National Stage of InternationalApplication No. PCT/EP2013/000994, filed Apr. 3, 2013, which designatesthe U.S. and was published by the International Bureau in English onOct. 10, 2013, and which claims the benefit of European Application12002402.1, filed Apr. 3, 2012; the contents of all of which are herebyincorporated herein in their entirety by reference.

FIELD OF THE INVENTION

The present invention relates to a process of transiently transfecting aplant or leaves on a plant. The invention also relates to a process oftransiently expressing a DNA sequence of interest in a plant or inleaves on a plant. Further, the invention relates to an Agrobacteriumstrain.

BACKGROUND OF THE INVENTION

Current genetic engineering processes for agriculture are all based onstable genetic modification of crop species, demonstrated first in 1983(Fraley et al 1983; Barton et al 1983) and commercialized since 1996.Although agriculture processes based on plant stable genetictransformation is a reality today and is a basis of successful newpractices, it has multiple limitations, the main ones being very longtime and high cost required for development of transgenic crops. Generalconsensus among the companies involved in plant biotechnology is thatthe R&D process requires, depending on the crop species, between 8 and16 years, and the total average development cost is estimated to bebetween $100 and $150 million. Because of these limitations, after morethan 25 years since the discovery of a plant genetic transformationprocess, only a handful traits and few GM crop species have beencommercialized thus far.

It is known that plant cells and whole plants can also be re-programmedtransiently (i.e. without stable integration of new genetic material ona plant chromosome), and the transient processes, such as viralinfections, are fast. Such transient processes could in principle allowa very fast modification of plant metabolism in favor of certainproducts that are of interest to the user. Such processes require a DNAor RNA vector (a virus or a bacterium), that has been engineered toeffectively and safely transfect the plant. Earlier attempts to usevectors based on plant viruses have been partially successful in thatthey allow transfection of plants for manufacturing of high-valuerecombinant proteins such as certain biopharmaceuticals (Gleba et al2007, 2008; Lico et al 2008). Use of viruses for manipulation of othertraits, such as input traits (for example, herbicide resistance,Shiboleth et al 2001; Zhang and Ghabiral 2006) have been described inthe literature, but virus transfection introduces so many undesiredchanges in the infected host that this kind of transient process is notpursued anymore for input traits. Transient processes can also be builtaround the ability of Agrobacterium species to transfer part of their Tiplasmid to eukaryotic, in particular, plant cells. Use ofAgrobacterium-based transfection is a basis for genetic manipulationssuch as genetic transformation protocols and of laboratory transienttransfection assays. Industrial applications of Agrobacterium-basedtransfection have also been limited to recombinant proteinmanufacturing, because the optimal application conditions such as vacuuminfiltration of plants with bacterial suspensions cannot be used on alarge scale in the field, whereas spraying aerial parts or wateringplants with bacterial solutions results in a supposedly very smallproportion of plant cells to be transfected, and previous studies simplydid not address that specific question.

Agrobacterium tumefaciens and A. rhizogenes are broadly used in researchlaboratories worldwide for transient transfection and stable genetictransformation of plants. These applications are based on the ability ofAgrobacterium to transfer genetic information to eukaryotic cells. Manyof the transgenic plants cultivated today, such as soybeans, canola andcotton, have been generated through Agrobacterium-mediated genetictransformation. The essential difference between the transient andstable transformation is that in the process of stable transformation,Agrobacterium-delivered DNA is eventually integrated into a plantchromosome, and is afterwards inherited by the plant progeny. Suchintegration events are rare even in laboratory experiments specificallydesigned to provide massive contacts between plant cells and bacteria:thus for the selection of stable transformants, specific selectivescreening methods have to be utilized and specific plant explants (richin meristematic tissues) selected for optimum transformation andregeneration into whole plants are employed. Subsequently, the knowledgeaccumulated in this science domain is of limited value to thoseinterested in transient processes where many cells of the plant bodyshould be affected without selection for transfected cells.

Transient transfection, on the other hand, takes into account onlyearlier steps of Agrobacterium-driven DNA delivery into a nucleus of aplant cell, along with the fact that such delivered DNA molecules can betranscribed in a nucleus even in the absence of DNA integration into aplant chromosome, such expression resulting in a transient metabolicreprogramming of a plant cell. Such reprogramming has been developedinto a laboratory tool for rapid evaluation of different geneticexperiments. Whereas there is considerable body of knowledge about.Agrobacterium-mediated DNA transfer to plant cells, that information isinvariably limited to laboratory scale experiments, and thus far, therewere very few attempts to develop industrial scale applicationsinvolving Agrobacterium, as a DNA vector.

One of the limitations of laboratory applications is the fact thatAgrobacterium-based DNA delivery requires certain treatments that aredifficult or impossible to apply in open field or on a large scale. Intypical transient experiments, cultured plant cells or parts of plants(explants) are treated with an excess of bacteria to provide for maximumdelivery. In typical research experiments, one is also interested inexpression levels that are not economically viable if done on anindustrial scale. In general, the research done in this domain has ledthe inventors to the conclusion that the parameters seriously affectingtransient expression are those allowing for the best interaction accessof agrobacteria to plant cells within a plant body. Most such studiesutilize vacuum infiltration, injection into plant leaf or surfactanttreatment, wounding of plant surface e.g. with razor blades, orcombination thereof. In fact, the only group that is developing anAgrobacterium-based transfection process for commercial production ofrecombinant proteins that does not involve further (virus-based)amplification of the original DNA, is the group of Medicago (D'Aoust etal 2008, 2009: Vezina et al, 2009). Their process relies entirely onvacuum infiltration as a delivery method. However, because of beingbased on great excess of bacteria to plant cell ratio, currentlaboratory protocols used for transient transfection of plants do nothave serious translational value, i.e. they cannot be directlyreplicated on an industrial level. Except in few cases (e.g. Vaquero etal, 1999, D'Aoust et al, 2008, 2009) they also have not addressedquantitatively the issue of efficiency of the transient transfectionprocess. (Examples of such research are multiple, we provide a citationfor just a few representative ones: Li et al, 1992; Liu et al, 1992;Clough and Bent, 1998; De Buck et al, 1998, 2000; Chung et al, 2000;Yang et al, 2000; Zambre et al, 2003; Wroblewski et al, 2005; Lee andYang, 2006; Zhao et al, 2006; Shang et al, 2007; Jones et al., 2009; Liet al, 2009; De Felippes and Weigel, 2010).

One of the industrial processes being under development today ismagnifection, a process that is based on vacuum-infiltration ofagrobacteria into leaves of plants. The magnifection process(trademarked by Icon Genetics GmbH as magnICON® and covered by severalpatents/patent applications) is a simple and indefinitely scalableprotocol for heterologous protein expression in plants, which is devoidof stable genetic transformation of a plant, but instead relies ontransient amplification of viral vectors delivered to multiple areas ofa plant body (systemic delivery) by Agrobacterium as DNA precursors.Such a process is in essence an infiltration of whole mature plants witha diluted suspension of agrobacteria carrying T-DNAs encoding viral RNAreplicons. In this process, the bacteria assume the (formerly viral)functions of primary infection and systemic movement, whereas the viralvector provides for cell-to-cell (short distance) spread, amplificationand high-level protein expression. The scale-up (industrial) version isbuilt around fully assembled viral vectors (rather than pro-vectorsrequiring in planta assembly) and requires apparatuses forhigh-throughput Agrobacterium delivery to whole plants by vacuuminfiltration. The process can be scaled up but it requires submersion ofaerial parts of plants into bacterial suspension under vacuum (theprocess involves inverting plants grown in pots or in trays), aprocedure that imposes limitations on the volumes of biomass that can betreated in this way, on the throughput of the process, on the ways theplants can be cultivated prior to treatment, and it also carries certaincosts that limit the use of the process to high-cost products, such asrecombinant biopharmaceuticals only. The magnifection process isefficient as it allows transfection of almost all leaf cells in treatedplants, or approximately 50% of the total aerial plant biomass (the restbeing stems and leaf petioles). The process has been optimized in manyways, see e.g. Marillonnet et al, 2005. However, the current process hasbeen built entirely around bacterial delivery methods such as injectioninto a plant leaf or vacuum-infiltration (e.g. Simmons et al, 2009),wounding of leaves (Andrews and Curtis, 2005), or pouring agrobacteriainto soil (‘agrodrenching’, Ryu et al, 2004; Yang et al, 2008), butthese methods can not be applied for the mass treatment of the plants ina field (reviewed in Gleba et al, 2004, 2007, 2008; Gleba & Giritch,2010, 2011; Lico et al, 2008; original articles of our group includeGiritch et at 2006; Marillonnet et al., 2004, 2005; Santi et at, 2006;and ideologically similar papers from other research groups—Voinnet etal, 2003; Sudarshana et al, 2006; Geo et al, 2006; Mett et al, 2007;Lindbo, 2007a,b; Plesha et al, 2007, 2009; Huang et al, 2006; Regnard etal 2009; Green et al, 2009; Shoji et al, 2009).

Attempts to use Agrobacterium treatment on whole plants (in planta)without vacuum-infiltration have resulted in a very low number ofinitially transfected cells, thus greatly limiting the practicalapplication of the process. Moreover, since no selection for transfectedplant cells is done in transient transfection systems, the entiretransient transfection process is of too low efficiency for large scaleapplications if vacuum-infiltration is to be avoided. Further, severalplant species such as soybean or rape seed are difficult to transfect byAgrobacterium, unless specific plant tissue is used, whereby in plantatransient transfection has not been achieved to a significant extent.

SUMMARY OF THE INVENTION

Departing from the prior art, it is an object of the present inventionto provide an efficient process of transient in planta transfection. Itis another object of the invention to provide an efficient process oftransiently expressing a DNA sequence of interest in planta. Further, itis an object of the invention to provide an efficient process allowingtransient plant transfection using Agrobacterium on a large (industrial)scale (i.e. to many plants in parallel) without the need for theapplication of pressure differences to introduce Agrobacterium into theintercellular space of plants. It is also an object to provide anAgrobacterium cell and strain suitable for this purpose.

These problems are solved by a process of transiently transfecting aplant or leaves on a plant, comprising contacting said plant or saidleaves with a suspension comprising Agrobacterium cells of strain CryXdeposited under accession No: DSM25686 or a derivative strain of strainCryX, wherein said derivative strain has the chromosomal background ofstrain CryX or said derivative strain contains the vir plasmid of strainCryX or a derivative of said vir plasmid.

Further provided is a process of transiently expressing a DNA sequenceof interest in a plant, comprising contacting said plant or said leaveson said plant with a suspension comprising Agrobacterium cells of strainCryX deposited under accession No: DSM25686 or a derivative strain ofstrain CryX, wherein said derivative strain has the chromosomalbackground of strain CryX or said derivative strain contains the virplasmid of strain CryX or a derivative of said vir plasmid.

The invention also provides Agrobacterium strain CryX having DSMaccession No: DSM25686 or a derivative strain of strain CryX, whereinsaid derivative strain has the chromosomal background of strain CryX, orsaid derivative strain contains the vir plasmid of strain CryX or aderivative of said vir plasmid; or an Agrobacterium cell of strain CryXor of said derivative strain.

The invention also provides Agrobacterium cells of strain CryX havingDSM accession No: DSM25686 or a derivative thereof, said cellscontaining a binary vector containing in T-DNA a DNA sequence ofinterest to be transfected into cells of a plant, wherein the binaryvector may encode a VirG protein from strain CryX or a closely relatedVirG protein as defined below.

The invention further provides a kit comprising:

-   -   an Agrobacterium cell of said strain or said derivative strain        as defined above and    -   a binary vector containing in T-DNA a DNA sequence of interest        to be transfected into cells of a plant.        The binary vector may encode a VirG protein from strain CryX or        a closely related VirG protein as defined below.

The invention also provides the vir plasmid of strain CryX andAgrobacterium cells having the chromosome of strain CryX.

The invention further provides an aqueous cell suspension ofAgrobacterium strain CryX having DSM accession No: DSM25686 or aderivative strain of strain CryX (as defined herein), said suspensionhaving a cell concentration of at most 1.1·10⁶ cfu/ml of the suspension,preferably at most 4.4·10⁶ cfu/ml of the suspension, and more preferablyof at most 1.1·10⁵ cfu/ml of the suspension.

The inventors of the present invention have found a way of stronglyincreasing the transient transfection efficiency of plants byAgrobacterium. The inventors have identified an Agrobacterium strain(Agrobacterium strain CryX) that achieves particularly high efficiencyin transient transfection in planta with a wide variety of plants.Notably, strain CryX achieves much higher transient transfectionefficiency in planta than other Agrobacterium strains that are used as astandard for plant transformation or transfection such as strain LBA4404or EHA105 (see page 64 of Slater et al., in: Plant Biotechnology, 2^(nd)edition, Oxford University Press, 2008). The inventors have furtherfound that strain CryX achieves higher transient transfection efficiencythan related Agrobacterium strains such as Chry5/KYRT1. Moreover, theinventors have found that a particularly high transfection efficiencycan be obtained when a virG gene, notably the virG gene fromAgrobacterium strain LBA4404 or a virG gene that is closely related tothat from LBA4404 is expressed in chrysopine or succinamopine-typeAgrobacterium tumefaciens strains.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show T-DNA regions with DNA sequences of interest ofvectors used in the examples. Pact2: promoter of Arabidopsis actin2gene; o: 5′-end from TVCV (turnip vein clearing virus); RdRp:RNA-dependent RNA polymerase open reading frame (ORF) from cr-TMV(crucifer-infecting tobamovirus); MP; movement protein ORF from cr-TMV;N: 3′-non-translated region from cr-TMV; Tnos or nos: nopaline synthaseterminator; white segments interrupting grey segments in the RdRp and MPORFs indicate introns inserted into these ORFs for increasing thelikelihood of RNA replicon formation in the cytoplasm of plant cells,which is described in detail in WO2005049839; GUS: coding sequence ofGUS protein; GFP: green fluorescent protein coding sequence; fs:frame-shift deleting cell-to-cell movement ability; P35S: 35S promoter;P19; gene silencing suppressor of tomato bushy stunt virus (cf. Plant J.33, 949-56); Tocs: ocs terminator; LB: left T-DNA border; RB: rightT-DNA border.

FIG. 2 shows a comparison of different Agrobacterium tumefaciens strainsfor their transient transfection efficiency. Photographs show GFPfluorescence 4 dpi (days post infection) under uv light due to TMV-basedGFP expression after syringe infiltration of Nicotiana benthamianaleaves with diluted agrobacterial cultures as described in Example 2.Numerals 10⁻², 10⁻³ and 10⁻⁴ show the concentration factors of theovernight agrobacterial cultures of OD=1.3 at 600 nm that correspond to10²-fold, 10³-fold and 10⁴-fold dilutions, respectively. The compositionof the buffer for infiltration is 5 mM MES, pH5.5 and 10 mM MgSO₄. Eachinfiltration was performed in triplicate using three independent leavesof the same plant.

(A) TMV-based vector capable of cell-to-cell movement: TMV(MP)-GFP(pNMD560).

(B) TMV-based vector lacking cell-to-cell movement ability:TMV(fsMP)-GFP (pNMD570).

1- Agrobacterium tumefaciens strain AGL1;

2- Agrobacterium tumefaciens strain EHA105;

3- Agrobacterium tumefaciens strain GV3101;

4- Agrobacterium tumefaciens strain ICF320;

5- Agrobacterium tumefaciens strain CryX;

6- Agrobacterium tumefaciens strain LBA4404;

7- Agrobacterium tumefaciens strain LBA9402.

FIG. 3 demonstrates the influence of virG gene overexpression ontransient transfection efficiency for AGL1, EHA105, ICF320 and GV3101strains, virG sequences from GV3101 and LBA4404 strains carrying theN54D mutation as well as native sequence from LBA4404 strain were usedfor comparison. Photographs show GFP fluorescence 4 (FIG. 3A) and 6(FIG. 3B) dpi under uv light due to TMV-based GFP expression aftersyringe infiltration of Nicotiana benthamiana leaves of the same agefrom 3 independent plants with diluted agrobacterial cultures asdescribed in Example 3. Numerals 10⁻², 10⁻³ and 10⁻⁴ indicate thefactors by which the cell concentrations of the overnight agrobacterialcultures of OH=1.3 at 600 nm were reduced. Thus, the factors 10⁻², 10⁻³and 10⁻⁴ correspond to 10²-, 10³- and 10⁴-fold dilutions, respectively.The composition of the buffer for infiltration; 5 mM MES, pH5.3 and 10mM MgCl₂. In all cases TMV-based vectors capable of cell-to-cellmovement were used.

1- pNMD560 (no virG) in GV3101;

2- pNMD064 (virGN54D/GV3101) in GV3101;

3- pNMD063 (virGN54D/LBA4404) in GV3101;

4- pNMD062 (virG/LBA4404) in GV3101;

5- pNMD560 (no virG) in ICF320;

6- pNMD064 (virGN54D/GV3101) in ICF320;

7- pNMD063 (virGN54D/LBA4404) in ICF320;

8- pNMD062 (virG/LBA4404) in ICF320;

9- pNMD560 (no virG) in EHA105;

10- pNMD064 (virGN54D/GV3101) in EHA105;

11- pNMD063 (virGN54D/LBA4404) in EHA105:

12- pNMD062 (virG/LBA4404) in EHA105;

13- pNMD560 (no virG) in AGL1;

14- pNMD064 (virGN540/GV3101) in AGL1;

15- pNMD063 (virGN54D/LBA4404) in AGL1;

16- pNMD062 (virG/LBA4404) in AGL1.

FIGS. 4A and 4B show the influence of virG gene overexpression on thetransient transfection efficiency of GV3101 and CryX strains. virGsequences from GV3101 and LBA4404 strains carrying N54D mutation as wellas native sequence from LBA4404 strain were used for comparison.Photographs show GFP fluorescence 3, 4 and 5 dpi under uv light due toTMV-based GFP expression after the syringe infiltration of Nicotianabenthamiana leaves of the same age from 3 independent plants withdiluted agrobacterial cultures as described in Example 3. Numerals 10⁻⁴and 10⁻⁵ indicate the concentration factors of the overnightagrobacterial cultures of OD=1.3 at 600 nm. The composition of thebuffer for infiltration; 5 mM MES, pH5.3 and 10 mM MgCl₂. TMV-basedvectors capable of cell-to-cell movement were used.

1- pNMD560 (no virG) in GV3101 strain;

2- pNMD064 (virGN54D/GV3101) in GV3101 strain;

3- pNMD063 (virGN54D/LBA4404) in GV3101 strain;

4- pNMD560 (no virG) in CryX strain;

5- pNMD064 (virGN54D/GV3101) in CryX strain;

6- pNMD063 (virGN54D/LBA4404) in CryX strain.

FIG. 5 shows a comparison of transient transfection efficiencies forCryX and GV3101 strains in a range of dilutions of overnightagrobacterial cultures from 10⁻³ to 10⁻⁷ using syringe infiltration ofNicotiana benthamiana leaves. Numerals 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶ and 10⁻⁷indicate the concentration factors of the overnight agrobacterialcultures of OD=1.3 at 600 nm. These correspond to 10³, 10⁴, 10⁵, 10⁶ and10⁷-fold dilutions, respectively. The composition of the buffer forinfiltration: 5 mM MES, pH5.3 and 10 mM MgCl₂ in water. Photographs aretaken at 4 dpi.

1- pNMD560 (no virG) in GV3101 strain;

2- pNMD064 (virGN54D/GV3101) in GV3101 strain;

3- pNMD063 (virGN54D/LBA4404) in GV3101 strain;

4- pNMD560 (no virG) in CryX strain;

5- pNMD064 (virGN54D/GV3101) in CryX strain;

6- pNM0063 (virGN54D/LBA4404) in CryX strain.

FIG. 6 shows the results of testing different Agrobacterium strains fortransient transfection of soybean using spraying with suspension ofagrobacterial cells. pNMD2190 construct (35S:GUS; 35S:p19 andvirGN54D/LBA4404 in the plasmid backbone) was used with AGL1, EHA105,CryX and LBA4404 strains; pNMD2180 construct (35S:GUS; 35S:p19 andvirGN54D/GV3101 in the plasmid backbone) was used with GV3101 and ICF320strains. Staining of leaves for the GUS activity was performed at 11dps.

(A) For spraying, liquid Agrobacterium cultures of OD₆₀₀=1.3 werediluted in the ratio 1:10 with buffer containing 5 mM MES, pH5.3; 10 mMMgCl₂ and 0.05% (v/v) Tween®20.

(B) For spraying, liquid Agrobacterium cultures of OD₆₀₀=1.3 werediluted in ratios 1:10, 1:100 and 1:1000 with buffer containing (5 mMMES, pH5.3; 10 mM MgCl₂ and 0.05% (v/v) Tween®20) supplemented withsilicon carbide particles of size 800 in the concentration 0.3% (w/v).

FIG. 7 shows test results of CryX and EHA105 strains for transienttransfection of cotton Gossipium hirsutum L. using spraying withsuspension of agrobacterial cells. For spraying, liquid Agrobacteriumcultures of OD₆₀₀=1.3 were diluted in the ratio 1:10 with buffercontaining 5 mM, MES pH5.3; 10 mM MgCl₂ and 0.25% (v/v) Silwet L-77. Fortesting, constructs pNMD1971 (35S:GUS; 35S:p19), pNMD2180 (35S:GUS;35S:p19 and virGN54D/GV3101 in the plasmid backbone) and pNMD2190(35S:GUS; 35S:p19 and virGN54D/LBA4404 in the plasmid backbone) wereused. GUS activity test was performed at 6 dps.

FIG. 8 shows a comparison of two accessions of Agrobacterium tumefaciensChry5/KYRT1 strains received from different laboratories using aTMV-based vector capable of cell-to-cell movement (TMV-GFP, pNMD560vector). Photographs show GFP fluorescence 4 dpi (days post infection)under uv tight due to TMV-based GFP expression after syringeinfiltration of Nicotiana benthamiana leaves with diluted overnightagrobacterial cultures of OD=1.3 at 600 nm as described in Example 8.The strain obtained from the laboratory of Dr. G. Collins in theUniversity of Kentucky (Lexington, USA) is infiltrated on the right-handside of each leaf. The accession from the Institute of Cell Biology andGenetics Engineering (ICBGE, Kiev, Ukraine), is infiltrated on theleft-hand side of each leaf. The composition of the buffer forinfiltration is 5 mM MES, pK5.5 and 10 mM MgSO₄. Each infiltration wasperformed in triplicate using three independent leaves of the sameplant.

1- ICBGE accession, concentration factor 10⁻¹ (10-fold dilution);

2- ICBGE accession, concentration factor 10⁻²;

3- ICBGE accession, concentration factor 10⁻³;

4- Kentucky University accession, concentration factor 10⁻¹;

5- Kentucky University accession, concentration factor 10⁻²;

6- Kentucky University accession, concentration factor 10⁻³.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, a particular class of Agrobacteriumtumefaciens strains is used for transient transfection of plants such asleaves on a plant. This class of Agrobacterium comprises A. tumefaciensstrain CryX and derivative strains thereof as defined below. Strain CryXwas deposited with DSMZ-Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH, Inhoffenstraβe 7B, 38124 Braunschweig, Germany onFeb. 23, 2012 under the Budapest Treaty. Accession number DSM25686 hasbeen assigned to it. Strain CryX has a chromosomally integratedrifampicin resistance.

Strain CryX is related to the Chrysanthemum morifolium-derivedAgrobacterium strain Chry5 that has been identified by Busch & Puepke in1991. It has been shown in their paper that the strain is a biotype I bytraditional biotype tests and that it produces tumors on at least 10plant species. It has been characterized as unusual because of itsability to form efficiently large tumors on soybean (Glycine max) andfor this reason, it has been subsequently further characterized in anumber of papers by various groups. Chry5 is unable to utilize octopineor mannopine as a carbon source; instead it is able to catabolize asingle isomer each of nopaline and succinamopine, at the same time it isinsensitive to agrocin 84 (Busch & Puepke, 1991). In addition,Chry5-strain-induced tumors produce a family of Amadori-type opines thatincludes deoxyfructosyl glutamine (Dfg) and its lactone, chrysopine(Chy) (Palanichelvam et al., 2000). The isolates of Chry5 have beenshown to contain at least two plasmids, one with a homology with pTiB6.Torisky et at (1997) have partially disarmed the strain by removingapprox. 16.5-kb segment from the 285-kb Ti plasmid of Chry5, includingapprox. 4 kb of the oncogenic T-DNA, through homologous recombination.This deletion mutant, named KYRT1, has been shown to be an efficientvector organism, and this partially disarmed derivative of Chry5 hassince been used by some researchers. More recently, Palanichelvamet etal. (2000) have developed a fully disarmed derivative.

In research that led to the present invention, the inventors haveinitially tested two accessions of Chry5/KYRT1 received from differentlaboratories. The strain obtained from the laboratory of Dr. G. Collins(Torisky et al., 1997) did not show any superiority over standardcomparator strains EHA105 and GV3101 in our transient studies and wasexcluded from further studies. An accession from the Institute of CellBiology and Genetics Engineering (Kiev, Ukraine), on the other hand, hasbeen found to be unusually active in its transient transfection andexpression efficiency and has been used in the present invention. Thislatter accession was deposited under the Budapest treaty in the officialdepository DSMZ-Leibniz-Institut Deutsche Sammlung von Mikroorganismenund Zelikulturen GmbH, Braunschweig, Germany under the name CryX, toreflect the fact that there is no clear provenance information on it.

The original and subsequent papers have characterized the Chry5 strainin more detail. These studies aimed at standard characterization ofmolecular biology and genetics of the strain, as well as its comparativeability to induce tumors, to cause genetic transformation of differentplant species, as well as its ability to cause transient expression inAgrobacterium-treated explants. Results of these studies are brieflysummarized below.

Methods of Comparison Used to Characterize Agrobacterium tumefaciensChry5

1. Data on Oncogenicity

In the original paper of Bush & Puepke (1991), it has been establishedthat the Chry5 strain is able to cause tumors on 10 plant speciesrepresenting 7 plant families. The test involved semi-quantitativeevaluation of the number of plants with tumors caused by this strainversus the common laboratory strain B6. There were no significantdifferences in tumorigenicity between the strains in 6 out of 9 species(beets, kalanchoe, marigold, sunflower, tobacco and tomato). On collard,Chry5 has been approx. two times more efficient, and on soybean—approx.three times more efficient, whereas on pea, it was somewhat lessefficient than B6. Torisky et al, (1997) provided additional data ontumor formation on stems of tobacco and tomato; in this study, the Chry5strain and its partially disarmed derivative KYRT1 have been comparedwith two other Agrobacterium strains often used in transformationstudies, including A281, a succinamopine-type strain containing Bo542 Tiplasmid in the C58 chromosomal background and its disarmed derivativeEHA105 strain. It has been shown in that study that whereas the originalChry5 and the other succinamopine strain used, A281, are both highlytumorigenic, the partially disabled derivative KYRT1 and EHA105 were notactive.

2. Data on Transformation Efficiency using Partially Disabled and FullyDisabled Strains

Torisky et al. (1997) demonstrated that KYRT1 successfully transfers thebeta-glucuronidase (GUS) gene into tobacco leaf explants, producingGUS-expressing callus which could be regenerated into viable plants. Inthese experiments, the transformation efficiency of KYRT1 strain wasapproximately the same as was shown for EHA105. Grant et al. (2003)found the KYRT1 strain to be on average threefold more efficient thanAGL 1 for producing transgenic plants of pea using for evaluationcotyledonary explants of three different plant genotypes.

In the work of Palanichelvam et al. (2000), it has been shown that KYRT1derivative is only partially disarmed and contains all of oncogenicT-right and the fragment of T-left regions. A Chry5 derivative withcompletely disarmed Ti plasmid, pKPSF2 (Palanichelvam et al., 2000),was, however, less efficient for the stable transformation of soybean,as the KYRT1 strain retains some hormonal effect on plant explantsenhancing somatic embryogenesis in soybean (Ko et al., 2004),

3. Data Characterizing Transient Activity

Again, Torisky et al (1997) were the first to study transient expressionof β-glucuronidase transgene caused by the Chry5 derivative KYRT1 byusing a quantitative assay of GUS expression in cotyledonary nodeexplants of soybean. These data indicated that KYRT1 derivative wasapprox. 2.5 times more efficient in causing transient expression whencompared to EHA105 or GV3850. KYRT1 was on average 2.8-fold moreefficient than EHA105 and C58C1 for producing transient β-glucuronidase(GUS) gene (gus) expression on cotyledonary petioles of a recalcitrantlegume plant, lentil (Lens culinaris M.) (Akcay et al., 2009), Akbulutet al. (2008) have measured GUS activity in explants derived fromwounded seedlings after treatment with KYRT1 and two other commonvectors, C58C1 and EHA105. The quantitative evaluation of GUS expressingspots has shown that after 16 hours of imbibition, there are nostatistically significant differences between KYRT1 and C58C1, and after40 hours, KYRT1 is better by ca. 50%, but only in one of two measurementpoints.

Interpretation of the above mentioned data in its entirety is difficultbecause different authors used different plant species, different plantexplants, different strains of Agrobacterium for comparison, and havemade their conclusions based on three different activity methods:tumorigenic activity, efficiency of genetic transformation andefficiency of transient expression. The process of interaction of aplant cell and an Agrobacterium is very complex, and it involvestransfer of T-DNA-protein complex, transfer of proteins such as VirE2(via a separate secretion system), transient expression of T-DNA genesin a plant cell, hormonal effect of expressed genes, integration of someT-DNA molecules into a plant chromosomal DNA, etc. Any of theseintermediate processes can influence the end result; therefore, data ontumorigenicity and on transformation efficiency gives no informationwith regard to the efficiency of T-DNA transfer and transientexpression. The presented data on transient activity are, on the otherhand, limited and the slight differences observed are inconclusive ornot practically relevant.

A major difference in the processes and strains described herein and themethods used in the prior art described above is the different biologyof the plant material used for transient expression studies. Whereas allprevious authors used in vitro cultured or excised plant explants richin meristematic tissues (the ultimate goal being ability to transform aplant cell and to regenerate a whole plant from said transgenic cell),such as excised embryo, parts of young seedlings, etc., the presentinvention relates to transient transfection of intact, developed plantswhich interact with Agrobacterium differently. On entire plants,agrobacteria enter the leaf via stomata (that is absent in other organsand in meristematic tissues) and not via wounds on plant explants. Asmentioned before, all authors without exception were using high bacteriadensities when treating plant explants.

Strain CryX of the present invention contains a vir plasmid that is atleast partially disarmed. “Disarmed” means that the vir plasmid and itshost Agrobacterium is not oncogenic, i.e. it does not insert oncogenesor genes for the production of opines into plant cells, either becauseit does not contain such genes or it cannot transfer such genes. A virplasmid comprises the vir genes (virulence genes) required for T-DNAtransfer into plant cells. Vir genes and their functions in T-DNAtransfer are known in the art and are e.g. summarized in the book ofSlater et al., Plant Biotechnology, 2^(nd) edition; Oxford UniversityPress, 2008; see also Hellens et al., Trends in Plant Science 5 (2000)446-451.

Strain CryX of the present invention is a binary strain, i.e. the virgenes required for transfer of T-DNA into plant cells and the T-DNA areon separate plasmids (see e.g. the book of Slater et al and the articleof Hellens et al. regarding binary Agrobacterium strains and vectorsystems). In the context of a binary Agrobacterium strain, the plasmidcontaining the vir genes is referred to herein as “vir plasmid” or “virhelper plasmid”. The plasmid containing the T-DNA to be transfected isreferred to as “vector” or “binary vector”. The term “strain” or“Agrobacterium strain” relates to components of the Agrobacterium otherthan the binary vector. Thus, herein, a binary Agrobacterium strain notcontaining a binary vector and after introduction of a binary vector arereferred to by the same strain name. Deposited strain CryX contains avir plasmid, but does not contain a binary vector.

The invention also relates to derivative strains of strain CryX. In anembodiment (i), a derivative strain of strain CryX has the chromosomalbackground of strain CryX. It may have the same chromosome as CryX inanother embodiment (ii), a derivative strain of strain CryX contains thevir plasmid of strain CryX. In a further embodiment (iii), a derivativestrain of strain CryX contains a derivative of the vir plasmid of strainCryX, and may have the chromosome of strain CryX. In embodiment (ii),the strain is a binary strain and is used in the processes of theinvention after introduction of a binary vector containing a T-DNA ofinterest. In embodiments (i) and (iii), the strains may be binarystrains. If they are binary strains, they are used in the processes ofthe invention after introduction of a binary vector containing a T-DNAof interest. Alternatively, in embodiments (i) and (iii), the T-DNA tobe transferred into plant cells in the processes of the invention may beinserted into the vir plasmid, such that the vir genes and the T-DNA arepresent on one and the same plasmid molecule. However, it is generallymore convenient and thus preferred to use binary strains.

The term “chromosomal background” is a standard term in the art ofAgrobacterium transformation or transfection (cf. Hellens et al., Trendsin Plant Science 5 (2000) 446-451). It relates to genetic material ofsaid Agrobacterium strain other than the Ti plasmids, vir helperplasmids and binary vectors. In one embodiment, a derivative strain ofstrain CryX has the same chromosome as strain CryX. A derivative strainhaving the chromosomal background of strain CryX may differ from CryX,for example, in the vir plasmid compared to the vir plasmid of CryX.Thus, a derivative strain of strain CryX may contain a vir plasmid thatis a derivative of the vir plasmid of strain CryX.

Whether a given Agrobacterium strain that has a chromosome that isnon-identical to that of strain CryX has a chromosomal background ofstrain CryX can be tested experimentally by comparing the T-DNA transferefficiency (or transfection efficiency) from a T-DNA-containing binaryvector of strain CryX with the strain to be tested that contains the virplasmid of CryX and the same binary vector. The strain to be tested isconsidered having the chromosomal background of CryX if it achieves atleast 70%, preferably at least 80% of the T-DNA transfer efficiency ofstrain CryX. Transfection efficiencies can be determined as described inExample 2. Alternatively, transfection efficiencies can be determined asin Example 2 but using a binary vector encoding a TMV-viral replicon notcapable of cell-to-cell movement such as pNMD570. T-DNA transferefficiency can also be determined by protoplast counting as described inWO 2005049839. In one embodiment, the chromosome of an Agrobacteriumstrain having the chromosomal background of strain CryX has a chromosomethat is the same in base sequence as that of strain CryX.

A derivative of the vir plasmid of strain CryX achieves, when present inAgrobacterium cells having the same chromosome as strain CryX, a similarefficiency of T-DNA transfer into plant cells from a T-DNA-containingbinary vector present in these cells. For this purpose, the derivativevir plasmid has a vir region sufficiently similar to that of the virplasmid of strain CryX. The derivative vir plasmid may encode the virGprotein of strain CryX or a closely related virG protein. Preferably,the derivative vir plasmid contains the virG gene of strain CryX. In oneembodiment, the derivative vir plasmid contains genes encoding at leasttwo of the following virulence proteins of CryX: VirA, VirG,VirB1-BirB11, VirC1, VirD1, VirD2, VirD4, VirE1, VirE2, VirF and VirJ.In a further embodiment, a derivative vir plasmid contains at least thegenes encoding the following virulence proteins of CryX: VirA, VirG,VirD2, and VirE2. In a further embodiment, a derivative vir plasmidcontains the entire vir region, i.e. all vir genes of the vir plasmid ofstrain CryX. The derivative vir plasmid may differ in the plasmidbackbone from the vir plasmid of CryX. For example, the derivative virplasmid may have a different or additional selective marker gene or mayhave deleted further nucleic acid portions from outside the vir region.In one embodiment, the derivative vir plasmid is pKYRT1 (U.S. Pat. No.5,929,306), notably if the derivative strain has the same chromosome asCryX.

Whether a given vir plasmid is a derivative vir plasmid in the sense ofthe present invention may be tested experimentally by comparing theT-DNA transfer efficiency from a T-DNA-containing binary vector betweenAgrobacterium of strain CryX and Agrobacterium having the chromosome ofstrain CryX but the vir plasmid to be tested under otherwise identicalconditions. In one embodiment, the Agrobacterium containing a derivativeplasmid according to the invention achieves at least 70%, preferably atleast 80%, more preferably at least 90% of the TDNA transfer efficiencyof strain CryX. Transfection efficiencies can be determined as describedin Example 2 and as mentioned above.

“Closely related virG protein” to the virG protein of CryX means a virGprotein that differs from the virG protein of CryX in at most 3non-conservative amino acid substitutions or in at most 6, preferably atmost 3 conservative amino acid substitutions. The non-conservative aminoacid substitutions may be at positions corresponding to positions 6, 7or 106 of the amino add sequence of SEQ ID NO: 1 which is the VirGprotein from Agrobacterium strain LBA4404, all other amino acid residuesbeing as in SEQ ID NO:1. In addition, the closely related virG proteinmay have an asparagine to aspartate substitution at the positioncorresponding to position 54 of SEQ ID NO: 1. The conservative aminoacid substitutions may be at positions 6, 7 and/or 106 of the amino acidsequence of SEQ ID NO: 1.

Herein, conservative substitutions are substitutions of amino acidresidues within each of the following four groups:

-   -   Ala, Pro, Gly, Glu, Asp, Gin, Asn, Ser, Thr    -   Val, Ile, Leu, Met    -   Lys, Arg, His    -   Phe, Tyr, Trp        All other amino acid residue substitutions are considered        non-conservative.

Herein, a “T-DNA of interest” is a DNA containing, between T-DNA leftand right border sequences, a DNA sequence of interest. A T-DNA ofinterest may be present or may have been incorporated by sub-cloninginto a vir plasmid such as the vir plasmid of strain CryX. In theprocesses of the invention, it is preferred to use a binary vectorsystem. Therefore, a T-DNA of interest is preferably present or willhave been incorporated into into a binary vector.

The binary vector to be used in the present invention is a DNA moleculecomprising a DNA sequence of interest to be transfected into plantcells. The DNA sequence of interest typically encodes a protein or anRNA to be expressed in cells of the transfected plants. The binaryvector is generally produced by inserting or cloning a nucleic acidconstruct containing the DNA sequence of interest into a cloning sitewithin T-DNA of a precursor binary vector, as generally done inAgrobacterium-mediated plant transfection. After said insertion, thenucleic acid construct is flanked by T-DNA left and right bordersequences for allowing transfection of said plant with said T-DNA. Inthe T-DNA of the binary vector, the DNA sequence of interest is presentsuch as to be expressible in plant cells. For this purpose, the DNAsequence of interest is, e.g. in said nucleic acid construct, typicallyunder the control of a promoter active in plant cells. Examples of theDNA sequence of interest are a DNA sequence encoding a DNA viralreplicon or an RNA viral replicon or a gene to be expressed. The genemay encode an RNA of interest or a protein of interest to be expressedin cells of the plant(s). Also the viral replicons typically encode anRNA or a protein of interest to be expressed in plants. The DNAconstruct may comprise, in addition to the DNA sequence of interest,other sequences such as regulatory sequences for expression of the DNAsequence of interest. Binary vectors usable in the invention are knownto the skilled person, e.g. from the references cited in theintroduction or from text books on plant biotechnology such as Slater,Scott and Fowler, Plant Biotechnology, second edition, Oxford UniversityPress, 2008. The binary vector typically has an antibiotic resistancegene for allowing selection in bacteria such as E. coli.

For increasing transfection efficiency, the binary vector may comprise,outside the T-DNA, a virG gene expressible in said Agrobacterium strain.Alternatively, an additional plasmid may be inserted into saidAgrobacterium strain, whereby said additional plasmid contains a virGgene expressible in said Agrobacterium strain (Pazour et al., Proc.Natl. Acad. Sci. USA 88 (1991) 6941-6945). The virG gene preferablyencodes a VirG protein from Agrobacterium tumefaciens strain LBA4404 ora closely related VirG protein. Further, the VirG protein may have theN54D mutation at the position corresponding to position 54 of SEQ ID NO:1, i.e. the VirG protein from A. tumefaciens strain LBA4404. The N54Dmutation in a VirG protein from another Agroabacterium strain wasdescribed by Jung et al., Current Microbiology 49 (2004) 334-340.

The closely related VirG protein may be

(i) a protein comprising at least 235, preferably at least 239consecutive amino acids of the amino acid sequence of SEQ ID NO: 1 or ofthe amino acid sequence of the N54D mutant of the amino acid sequence ofSEQ ID NO: 1; or

(ii) a protein comprising an amino acid sequence having not more than 3non-conservative amino acid substitutions and not more than 10conservative amino acid substitutions of the amino acid sequence of SEQis NO: 1 or the N54D mutant thereof; or

(iii) a protein comprising an amino acid sequence having not more than20, preferably not more than 10, conservative amino acid substitutionsof the amino acid sequence of SEQ ID NO: 1 or the N54D mutant thereof.

In items (ii) and (iii), said protein preferably has an asparagine oraspartate residue at the position corresponding to position 54 of SEQ IDNO: 1

Possible positions for the conservative amino acid substitutions in theembodiments mentioned above are positions 6, 7, 18, 35, 38, 42, 44, 66,69, 73, 81, 86, 89, 97, 106, 107, 122, 124, 133, 135, 143, 147, 150,165, 188, 208, 212, 213, 232, 235, and 238 of SEQ ID NO: 1, while aminoacid residues at other positions are those as in SEQ ID NO:1. Possiblepositions for non-conservative substitutions are positions 6, 7 and 106of the amino acid sequence of SEQ ID NO: 1.

In embodiments wherein strong expression of a protein or RNA is desiredor wherein accumulation of viral nucleic acids to high amounts in cellsof said plant and possible negative effects on plant health is not aconcern, the nucleic acid construct or DNA sequence of interest mayencode a replicating viral vector that can replicate in plant cells. Inorder to be replicating, the viral vector contains an origin ofreplication that can be recognized by a nucleic acid polymerase presentin plant cells, such as by the viral polymerase expressed from thereplicon. In case of RNA viral vectors, the viral replicons may beformed by transcription, under the control of a plant promoter, from theDNA construct after the latter has been introduced into plant cellnuclei. In case of DNA viral replicons, the viral replicons may beformed by recombination between two recombination sites flanking thesequence encoding the viral replicon in the DNA construct, e.g. asdescribed in WO00/17365 and WO 99/22003. If viral replicons are encodedby the DNA construct, RNA viral replicons are preferred. Use of DNA andRNA viral replicons has been extensively described in the literature atleast over the last 15 years. Some examples are the following patentpublications by Icon Genetics: WO2008028661, WO2007137788, WO2006003018,WO2005071090, WO2005049839, WO02097080, WO02088369, and WO02068664. Anexample of DNA viral vectors are those based on geminiviruses. For thepresent invention, viral vectors or replicons based on plant RNAviruses, notably based on plus-sense single-stranded RNA viruses arepreferred. Examples of such viral vectors are tobacco mosaic virus (TMV)and potexvirus X (PVX) used in the examples. Potexvirus-based viralvectors and expression systems are described in EP2061890. Many otherplant viral replicons are described in the patent publications mentionedabove.

When performing the process of the invention, the binary vectorcontaining in T-DNA the DNA sequence of interest may be introduced intothe Agrobacterium strain containing the vir plasmid or its derivative byconventional methods such as electroporation. A culture of the straincontaining the binary vector is then grown in suitable media, typicallyin the presence of a selective agent for selecting Agrobacterium cellscontaining the binary vector, and, optionally, sub-cultured to producethe desired amount of an aqueous suspension comprising the Agrobacteriumcells. The obtained suspension may be diluted to the desiredconcentration with water, a suitable buffer or media and be used fortransfecting a plant or leaves on the plant. Alternatively, if the T-DNAis part of the vir plasmid, the T-DNA containing vir plasmid isintroduced into Agrobacterium by conventional methods such aselectroporation and further treated as described for the binary system.

In the processes of the invention, in planta transfection is used. Inplanta means that the processes are performed on whole living plantsafter the seedling stage, preferably on fully developed plants, ratherthan on excised or in vitro cultivated plant tissues or organs.Preferably, the process is applied to many plants in parallel such asplants growing on a field.

Said plants may be contacted with the suspension of Agrobacterium cellsby infiltration with or without application of vacuum. In oneembodiment, notably when applied to multiple plants in parallel, theplants may be contacted with the suspension by spraying. The aqueoussuspension used in the processes of the invention may have aconcentration of Agrobacterium cells of at most 1.1·10⁹ cfu/ml, whichcorresponds approximately to an Agrobacterium culture in LB-medium of anoptical density at 600 nm of 1. Due to the high transfection efficiencyachieved in the invention, much lower concentrations may, however, beused, which allows treatment of many plants such as entire farm fieldswithout the need for huge fermenters for Agrobacterium production. Thus,the concentration is preferably at most 2.2·10⁷ cfu/ml, more preferablyat most 1.1·10⁷ cfu/ml, more preferably at most 4.4·10⁶ cfu/ml, in oneembodiment, the concentration is at most 1.1·10⁶ cfu/ml of thesuspension. In a further embodiment, the concentration is at most4.4·10⁵ cfu/ml of the suspension, and in a further embodiment, theconcentration is at most 1.1·10⁵ cfu/ml of the suspension

For avoiding determination of cell concentrations in terms of cfu/ml,concentrations of agrobacterial suspensions are frequently assessed bymeasuring the apparent optical density at 600 nm using aspectrophotometer. Herein, the concentration of 1.1·10⁷ cfu/mlcorresponds to a calculated optical density at 600 nm of 0.01, wherebythe calculated optical density is defined by a 100-fold dilution withwater or buffer of a suspension having an optical density of 1.0 at 600nm. Similarly, the concentrations of 4.4·10⁶ cfu/ml, 1.1·10⁶ cfu/ml,4.4·10⁵ cfu/ml and 1.1·10⁵ cfu/ml of the suspension correspond to acalculated optical density at 600 nm of 0.004, 0.001, 0.0004, and 0.0001respectively, whereby the calculated optical densities are defined by a250-fold, 1000-fold, 2600-fold, or 10000-fold, respectively, dilutionwith water or buffer of a suspension having an optical density of 1.0 at600 nm.

Thus, in a particularly preferred embodiment, the invention provides aprocess, and Agrobacterium cell suspension therefor, of transientlyexpressing a DNA sequence of interest in a plant, comprising contactingsaid plant or said leaves on said plant with a suspension comprisingAgrobacterium cells of strain CryX or a derivative strain of strainCryX, wherein said derivative strain has the chromosomal background ofstrain CryX or said derivative strain contains the vir plasmid of strainCryX or a derivative of said vir plasmid, wherein said suspension hasany of the maximum Agrobacterium cell concentrations mentioned in anyone of the preceding two paragraphs. In this embodiment, theAgrobacterium strain is preferably a binary strain containing a binaryvector comprises a virG gene expressible in said strain CryX or saidderivative strain. Said virG gene may encodes a VirG protein fromAgrobacterium tumefaciens strain LBA4404 of SEQ ID NO: 1, or is an N54Dmutant of the VirG protein encoded by the virG gene from A. tumefaciensstrain LBA4404.

It is possible to include an abrasive into the suspension for increasingthe transfection efficiency. The abrasive is a particulate material thatis essentially insoluble in the aqueous suspension of Agrobacteriumcells. The abrasive is believed to weaken, notably if used together witha wetting agent, the surface of plant tissue such as leaves, and therebyfacilitates penetration of Agrobacterium cells into the intercellularspace of plant tissue. Regarding possible abrasive usable in thepresence invention, particle sizes thereof, concentrations and possiblecommercial products, reference is made to International patentapplication published as WO 2012/019669 and disclosure regardingabrasives of this publication is incorporated herein.

The aqueous suspension of the invention may contain an agriculturalspray adjuvant. The spray adjuvant may be a surfactant or wetting agent.The surfactant and wetting agent has multiple advantages in the presentinvention. It reduces the surface tension of the water of the aqueoussuspension and makes the waxy surface of plant leaves more permeable foragrobacteria. It further improves the stability of the suspension andreduces settling of the abrasive in the suspension. Surfactants usablein the processes of the present invention are not particularly limited,and are disclosed in International patent application published as WO2012/019669. Preferred surfactants are nonionic surfactants of an HLBvalue of 12 or greater, preferably at least 13. As noninionicsurfactants, organo-silicone surfactants such aspolyalkyleneoxide-modified heptamethyltrisiloxane are most preferred inthe present invention. A commercial product is Silwet L77™ sprayadjuvant from GE Advanced Materials.

Surfactants such as those disclosed in WO 2012/019669 may be used singlyor in combination of two or more surfactants. Notably, the preferredorgano-silicone surfactants may be combined with other surfactants. Thetotal concentration of surfactants in the aqueous suspension of theinvention may be easily tested by conducting comparative sprayingexperiments, similarly as done in the examples. However, in general, thetotal concentration of surfactants may be between 0.005 and 2 volume- %,preferably between 0.01 and 0.5 volume- %, more preferably between 0.025and 0.2 volume- %, of said suspension. Since the density of surfactantsis generally close to 1.0 g/ml, the total concentration of surfactantsmay be defined as being between 0.05 and 20 g per liter of saidsuspension, preferably between 0.1 and 5.0 g, more preferably between0.25 and 2.0 g per liter of said suspension (including abrasive). If theabove organo-silicone surfactants such as polyalkyleneoxide-modifiedheptamethyltrisiloxane are used, the concentration of theorgano-silicone surfactant in the agrobacterial suspension used forspraying may be between 0.01 and 0.5 volume- %, preferably between 0.05and 0.2 volume- %. Alternatively, the concentration of theorgano-silicone surfactant in the agrobacterial suspension used forspraying may be defined as being between 0.1 and 5.0 g, preferablybetween 0.5 and 2.0 g per liter of said suspension.

In order to improve the physical properties of the aqueous suspension,it is possible to add highly dispersed sub-micron size silicic acid(silica) or porous polymers such as urea/formaldehyde condensate(Pergopak™). Notably, where the median particle size of the abrasive isbetween 0.1 and 30 μm, or in one of the preferred sub-ranges of thisrange given above, it is possible to add a highly dispersed sub-micronsize silica to the suspension. Herein, sub-micron size silica is silicahaving a median particle size between 0.01 and 0.5 μm, preferablybetween 0.02 and 0.5 μm, more preferably between 0.02 and 0.1 μm. Highlydispersed silicic acid such as Hi-Sil™ 233 (PPG Industries) cancontribute to the abrasive properties of the aqueous suspension (seeJensen et al., Bull. Org. mond. Sante, Bull. Wld Hlth Org. 41(1969)937-940). These agents may be incorporated in an amount of from 1to 10 g per liter of the suspension of the invention.

Further possible additives to the agrobacterial suspension are buffersubstances to maintain the pH of the suspension used for spraying at adesired pH, typically between 4.5 and 6.5, preferably between 5.0 and5.5. Further, inorganic soluble salts such as sodium chloride may beadded to adjust the ionic strength of the suspension. Nutrient brothsuch as LB medium may also be contained in the suspension.

The aqueous suspension for contacting with plants may be produced asfollows. In one method, the Agrobacterium strain or cells containing thedesired binary vector to be used in the process of the invention isinoculated into culture medium and grown to a high cell concentration.Larger cultures may be inoculated with small volumes of a highlyconcentrated culture medium for obtaining large amounts of the culturemedium. Agrobacteria are generally grown up to a cell concentrationcorresponding to an OD at 600 nm of at least 1, typically of about 1.5.Such highly concentrated agrobacterial suspensions are then diluted toachieve the desired cell concentration. For diluting the highlyconcentrated agrobacterial suspensions, water is used. The water maycontain a buffer. The water may further contain the surfactant of theinvention. Alternatively, the concentrated agrobacterial suspensions maybe diluted with water, and any additives such as the surfactant and theoptional buffer substances are added after or during the dilutionprocess. The abrasive may be added before, during or after dilution. Itis however preferred to agitate the suspension during addition of theabrasive to uniformly disperse the abrasive in the agrobacterialsuspension. The step of diluting the concentrated agrobacterialsuspension may be carried out in the spray tank of the sprayer used forspraying the diluted suspensions.

Said plants, notably leaves on said plant are then contacted with thesuspension of Agrobacterium cells to effect transient transfection ofcells of the plant. As explained above, contacting may be done byspraying. The sprayer to be used in the process of the invention mainlydepends on the number of plants or the area to be sprayed. For one or asmall number of plants to be sprayed, pump sprayers as widely used inhousehold and gardening can be used. These may have volumes of the spraytank of between 0.5 and 2 liters. For applications on a medium scale,manually operated hydraulic sprayers such as lever-operated knapsacksprayers or manually operated compression sprayers may be used. However,the high transfection efficiency achieved in the invention has its fullpotential in the transfection of many plants such as plants growing on afarm field or in a greenhouse. For this purpose, power-operatedhydraulic sprayers such as tractor-mounted hydraulic sprayers equippedwith spray booms can be used. Aerial application techniques usinghelicopters or airplanes are also possible for large fields. All thesetypes of sprayers are known in the art and are described for example inthe book “Pesticide Application Methods” by G. A. Matthews, thirdedition, Blackwell Science, 2000. In order to ensure a homogeneoussuspension in the spray tanks of the sprayers, small or medium sizesprayers may be shaken at regular intervals or continuously duringspraying. Large sprayers such as the tractor-mounted sprayers should beequipped with an agitator in the spray tank.

Considering the presence of agrobacterial cells and abrasive in thesuspensions to be sprayed, sprayers used in the invention should producespray of a droplet size at least of fine spray. Also, medium spray orcoarse spray in the classification of sprays used in “PesticideApplication Methods” by G. A. Matthews, third edition, BlackwellScience, 2000, page 74, may be used. The main purpose of the spraying inthe invention is wetting of plant tissue with the suspension. Thus, theexact droplet size is not critical. However, the transfection efficiencymay be further improved by providing the spray to plant surfaces withincreased pressure.

In the process of the invention, at least parts of plants are sprayed.In an important embodiment, plants growing in soil on a field aresprayed, i.e. plants not growing in movable pots or containers. Suchplants cannot be turned upside down and dipped into agrobacterialsuspension for vacuum infiltration. At least parts of plants are sprayedsuch as leaves. Preferably, most leaves are sprayed or entire plants.

The present invention is used for transient transfection of plants orfor transient with a DNA sequence of interest that may then betransiently expressed. The term “transient” means that the no selectionmethods are used for selecting cells or plants transfected with the DNAsequence of interest in the background of non-transfected cells orplants using, e.g. selectable agents and selectable marker genes capableof detoxifying the selectable agents. As a result, the transfected DNAsequence of interest is generally not stably introduced into plantchromosomal DNA. Instead, transient methods make use of the effect oftransfection in the very plants transfected.

The invention is generally used for transfecting multi-cellular plants,notably, higher plants. Both monocot and dicot plants can betransfected, whereby dicot plants are preferred. Plants for the use inthis invention include any plant species with preference given toagronomically and horticulturally important crop species. Common cropplants for the use in present invention include alfalfa, barley, beans,canola, cowpeas, cotton, corn, clover, lotus, lentils, lupine, millet,oats, peas, peanuts, rice, rye, sweet clover, sunflower, sweetpea,soybean, sorghum triticale, yam beans, velvet beans, vetch, wheat,wisteria, and nut plants. The plant species preferred for practicingthis invention include, but not restricted to, representatives ofGramineae, Compositeae, Solanaceae and Rosaceae.

Further preferred species for the use in this invention are plants fromthe following genera: Arabidopsis, Agrostis, Allium, Antirrhinum, Apium,Arachis, Asparagus, Atropa, Avena, Bambusa, Brassica, Bromus, Browaalia,Camellia, Cannabis, Capsicum, Cicer, Chenopodium, Chichorium, Citrus,Coffee, Coix, Cucumis, Curcubita, Cynodon, Dactylis, Datura, Daucus,Digitalis, Dioscorea, Elaeis, Eleusine, Festuca, Fragaria, Geranium,Glycine, Helianthus, Heterocallis, Hevea, Hordeum, Hyoscyamus, Ipomoea,Lactuca, Lens, Lilium, Linum, Lolium, Lotus, Lycopersicon, Majorana,Malus, Mangifera, Manihot, Medicago, Nemesia, Nicotiana, Onobrychis,Oryza, Panicum, Pelargonium, Pennisetum, Petunia, Pisurn, Phaseolus,Phleum, Poa, Prunus, Ranunculus, Raphanus, Ribes, Ricinus, Rubus,Saccharum, Salpiglossis, Secale, Senecio, Setaria, Sinapis, Solanum,Sorghum, Stenotaphrum, Theobroma, Trifolium, Trigonelia, Triticum,Vicia, Vigna, Vitis, Zea, and the Olyreae, the Pharoidese and others.

Preferably, the processes of the invention are applied to dicot plantsuch as Nicotiana benthamiana, tobacco, cotton, soybean, rapeseed,pepper, potato, or tomato.

In one embodiment, the process of the invention can be used forproducing a protein of interest in a plant or in many plants growing ona field. For this purpose, the plants may be sprayed with the suspensioncomprising the Agrobacterium cells containing the desired binary vectorat a desired growth state of the plants. If the main aim is to achievethe highest possible expression levels followed by harvesting plants forobtaining plant material containing high amounts of the protein, viralvectors may be used, since they generally give the highest expressionlevels.

In another embodiment, the process of the invention is used forgenerating or altering a trait in a plant such as an input trait. Inthis embodiment, excessive expression of a protein or RNA of interestmay not be desired for avoiding deleterious effects on plant health. Forsuch embodiments, non-replicating vectors (also referred to herein as“transcriptional vectors”), i.e. vectors lacking a functional origin ofreplication recognised by a nucleic acid polymerase present in the plantcells are preferred. Another application of the invention is RNAexpression, e.g. for RNA interference, wherein the interference signalcan spread in the plant from cells having expressed the signal to othercells. An example is the targeting of undesired viral DNA in plants asdescribed by Pooggin in Nat. Biotech. 21 (2003) 131. An example ofoncogene silencing that can be adapted to a transient system isdescribed by Escobar et al. Proc. Natl. Acad. Sci. USA 98 (2001)13437-13442. A further example is the control of coleopteran insectpests through RNA interference similar as described by Baum et al., Nat.Biotech. 25 (2007) 1322-1326 that can be adapted to the transientprocess of the invention by transiently transfecting pest-infestedplants with a DNA sequence of interest encoding the dsRNA such that itcan be expressed. Further methods applicable to the transient process ofthe invention are those described by Huang et al., Proc. Natl. Acad.Sci. USA 103 (2006) 14302-14306; Chuang et al., Proc. Natl. Acad. Sci.USA 97 (2000) 4985-4990.

Further, the process of the invention allows altering at a desired pointin time traits relating to the regulation of flowering time or fruitformation such as tuberisation in potato (Martinez-Garcia et al., Proc.Natl. Acad. Sci, USA 99 (2002) 15211-15216) or the regulation of theflavonoid pathway using a transcription factor (Deluc et al., PlantPhysical. 147 (2008) 2041-2053). Flowering may be induced by transientlyexpressing the movable florigen protein FT (Zeevaart, Current Opinion inPlant Biology 11 (2008) 541-547; Corbesier et al., Science 316 (2007)1030-1033). Parthenocarpic fruits in tomatoes may by produced on a largescale using the invention and the method described by Pandolfini et al.,BMC Biotechnology 2 (2002). Further applications of the invention are inthe context of altering cotton fiber development by way of MYBtranscription factors as described by Lee et al., Annals of Botany 100(2007) 1391-1401 or activation of plant defensive genes (Bergey et al.,Proc. Natl. Acad. Sd. USA 93 (1996) 12053-12058.

The invention also provides a process of protecting crop plants on afield from a pest. In such process, infestation of at least one of theplants from a plurality of plants growing in a lot or farm field may bedetermined. Due to the rapidness of the process of the inventionexpression of a protein or RNA detrimental to the pest needs to becaused only if infestation by the pest is determined. Thus, strong andconstitutive expression of pest toxins or dsRNA for RNAi even in theabsence of a risk of infestation is not necessary. Transient expressionof Bacillus thuringiensis endotoxins after the spraying with dilutedagrobacterial cultures harbouring corresponding PVX-based expressionvectors protected Nicotiana benthamiana plants from feeding damage bylarvae of the tobacco hornworm Manduca sexta (cf. FIG. 30 of WO2012/019660).

EXAMPLES Reference Example 1 Determination of Agrobacterium CellConcentration in Liquid Culture in Terms of Colony Forming Units (cfu)

The concentration of Agrobacterium cells in liquid suspension in termsof colony forming units per ml (cfu/ml) of liquid suspensions can bedetermined using the following protocol. Cells of Agrobacteriumtumefaciens strain ICF 320 transformed with construct pNMD620 were grownin 7.5 ml of liquid LBS medium containing 25 mg/L kanamycin (AppliChem,A1493) and 50 mg/L rifampicin (Carl Roth, 4163.2). The bacterial culturewas incubated at 28° C. with continuous shaking. Absorbance or opticaldensity of bacterial culture expressed in absorbance units (AU) wasmonitored in 1-ml aliquots of the culture using a spectrophotometer at600 nm wavelength (OD600). The cell concentration estimated as a numberof colony-forming units per milliliter of liquid culture (cfu/ml) can beanalyzed at OD600 values 1; 1.3; 1.5; 1.7 and 1.8. For this purpose 25μl aliquots of liquid culture were diluted with LBS-medium to achieve afinal volume of 25 ml (dilution 1:100). 2.5 ml of such 1:100 dilutionwere mixed with 22.5 ml of LBS to achieve the dilution 1:1000. Liquidculture dilutions 1:100; 1:1,000; 1:10,000; 1:100,000; 1; 1,000,000;1:10,000,000 and 1:100,000,000 were prepared similarly. Aliquots of lastthree dilutions were spread on agar-solidified LBS medium supplementedwith 26 mg/L kanamycin and 60 mg/L rifampicin (250 μl of bacterialculture per plate of 90 mm diameter). Plating of aliquots for eachdilution was performed in triplicate. After 2 days incubation at 28° C.,bacterial colonies were counted for each plate. Plating of 1:1,000,000and 1:10,000,000 dilutions resulted in few hundred and few dozencolonies per plate, respectively. So far as dilution 1:100,000,000provided just few colonies per plate, this dilution was not used forcalculation of cell concentration. The cell concentration was estimatedaccording to the formula: cfu/ml=4×number of colonies per plate×dilutionfactor.

For transforming cell concentrations as measured by absorbancemeasurements at 600 nm (in LB medium) and in terms of cell-formingunits, the following relation is used herein: an OD600 of 1.0corresponds to 1.1×10⁹ cfu/ml.

LBS Medium (Liquid)

1% soya peptone (papaic hydrolysate of soybean meal; Duchefa, S1330)

0.5% yeast extract (Duchefa, Y1333)

1% sodium chloride (Carl Roth, 9265.2)

dissolved in water, and the is adjusted to pH 7.6 with 1M NaOH (CarlRoth, 6771.2)

To prepare the solid LBS medium, liquid LBS medium was supplemented with1.5% agar (Carl Roth, 2266.2). Media were autoclaved at 121° C. for 20min.

Example 1: Vectors Used in the Following Examples

In this study we used TMV- and PVX-based viral vectors as well asstandard transcriptional vectors based on 35S CaMV promoter.

TMV-based vectors with cell-to-cell movement ability pNMD560, pNMD062,pNMD063 and pNMD064 (FIG. 1A) were created on the basis of vectorsdescribed in Marillonnet et at (2006). All these vectors are designedfor the expression of GFP as a reporter gene; they contain the insertionof a coding sequence of sGFP that is modified green fluorescent protein(GFP) from jelly fish Aequorea victoria (GeneBank accession no.EF030489). The pNMD560 construct contains, in sequential order, afragment from the Arabidopsis actin 2 (ACT2) promoter (GenBank accessionno. AB026654); the 5′ end of TVCV (GenBank accession no. BRU03387, basepairs 1-5455) and a fragment of cr-TMV [GenBank accession no. 229370,base pairs 5457-5677, both together containing 16 intron insertions]; asGFP coding sequence; cr-TMV 3′ nontranslated region (3′ NTR; GenBankaccession no. Z29370), and the nopaline synthase (Nos) terminator. Theentire fragment was cloned between the T-DNA left and right borders ofbinary vector.

The pNMD062 plasmid was created on the basis of pNMD560 construct. Forthis purpose, a DNA fragment comprising the coding sequence and a5′-upstream genomic region of a virG gene of octopine-type Ti-plasmidfrom LBA4404 strain of Agrobacterium tumefaciens (GenBank accession no.AF242881.1, base pairs 160603-161600) flanked by the sequence ctgtcgatcfrom the 5′-terminus and the sequence aagatcgacag (SEQ ID NO: 8) fromthe 3′ terminus was amplified by PCR and inserted into the plasmidbackbone using Afel restriction site.

The pNMD063 construct was identical to pNMD062 except for the N54Dmutation.

To create pNMD064 construct, a DNA fragment comprising the codingsequence containing the N54D mutation and 5′-upstream genomic region ofvirG gene of nopaline-type Ti-plasmid from GV3101 strain ofAgrobacterium tumefaciens (GenBank accession no. AE007871.2, base pairs194307-193333) was amplified by PCR and inserted into the plasmidbackbone of pNMD560 construct using Afel restriction site.

The pNMD570 construct (TMV-based vectors lacking cell-to cell movementability) was identical to pNMD560 except for a point mutation in theMP-coding sequence leading to an open reading frame shift that distortsMP translation (FIG. 1A).

The pNMD620 construct, a PVX-based vector without cell-to-cell andsystemic movement abilities for GFP expression, contained, in sequentialorder, a 35S CaMV promoter, coding sequences of the RNA-dependent RNApolymerase, triple gene block modules comprising 25 kDa, 12 kDa and 8kDa proteins, an sGFP coding sequence and a 3′-untranslated region. Theentire fragment was cloned between the T-DNA left and right borders ofbinary vector (FIG. 1B).

All transcriptional vectors were created on the basis of pICBV10, apBIN19-derived binary vector (Marillonnet et al., 2004, 2006). Theycontained two expression cassettes inserted within right and leftborders of the same T-DNA region (FIG. 1B). In the case of the pNMD1971construct, an expression cassette adjacent to the right bordercomprised, in sequential order, the Cauliflower mosaic virus (CAMV) 35Spromoter, omega translational enhancer from Tobacco Mosaic Virus, codingsequence of beta-glucuronidase from Escherichia coli (GenBank accessionno. 569414) containing the intron from Petunia hybrids PSK7 gene(GenBank accession no. AJ224165.1, base pairs 44114484), and theterminator from the nopaline synthase gene of Agrobacterium tumefaciens.The second expression cassette was inserted between the first one andthe T-DNA left border. It contained, in sequential order, theCauliflower mosaic virus (CAMV) 35S promoter, omega translationalenhancer from Tobacco Mosaic Virus, coding sequence of P19 suppressor ofsilencing from Tomato Bushy Stunt Virus (TBSV) (GenBank accession no.CAB56483.1) and terminator from octopine synthase gene of Agrobacteriumtumefaciens.

The pNMD2180 construct was created on the basis of the pNMD1971 vector.For this purpose, the Notl/Ndel fragment of the pNMD1971 construct wasreplaced with same fragment of pNMD064 construct containing virGN54Dgene of nopaline-type Ti-plasmid from GV3101 strain of Agrobacteriumtumefaciens flanked by 5′-upstream genomic region.

The pNMD2190 was created in a similar way. The Notl/Ndel fragment ofpNMD1971 construct was replaced with same fragment of pNMD063 vectorcontaining virGN54D gene of octopine-type Ti-plasmid from LBA4404 strainof Agrobacterium tumefaciens flanked by 5′-upstream genomic region.

Example 2: Strain CryX Shows Stronger Transient Transfection ofNicotiana benthamiana if Compared with Commonly Used Agrobacteriumtumefaciens Strains

We tested the number of Agrobacterium tumefaciens strains includingAGL1, EHA105, GV3101, ICF320, CryX, LBA4404 and LBA9402 for thetransient transfection of Nicotiana benthamiana plants. For this purposeplant leaves were infiltrated using a needleless syringe with 10⁻³ and10⁻⁴ dilutions of OD₆₀₀=1.3 of agrobacterial cultures of the sevenabove-mentioned strains harboring a GFP expression TMV-based vectorcapable of cell-to-cell movement (TMV-GFP, pNMD560 construct) as it isshown in FIG. 2A. In parallel, leaves were infiltrated with 10⁻² and10⁻³ dilutions of the overnight agrobacterial cultures of same strainscarrying TMV-based vector lacking cell-to-cell movement ability(TMV(fsMP)-GFP, pNMD570 construct) as it is shown in FIG. 2B.

Based on the density of fluorescing spots and the intensity of GFPfluorescence, we proved the efficient transient transfection for severalstrains (e.g., AGL1, EHA105, and LBA9402) however the transienttransfection efficiency of CryX strain was significantly higher for bothconstructs with all tested dilutions of agrobacterial cultures ifcompared with any other tested strain.

To provide a quantitative evaluation of transient transfectionefficiency for the CryX strain, we estimated the ratio between thenumber of cells in the bacterial suspension infiltrated in leaves andthe number of produced GFP fluorescent spots considered as a singletransfection event. For this purpose, leaves of 6-weeks old Nicotianabenthamiana plants were infiltrated using a syringe without needle with200 μl of agrobacterial cultures of OD600=1.3 diluted by dilutionfactors of 10⁻⁵, 10⁻⁵ and 10⁻⁷ with a buffer consisting of 5 mM MES, pH5.3 and 10 mM MgCl₂. CryX, EHA105, GV3101 and ICF320 agrobacterial cellscarried constructs pNMD560 (TMV-GFP vector). For the scoring ofbacterial cells, 100 μl aliquots of bacterial suspensions used for leafinfiltration were plated in triplicate on LB-agar plates containing 50μl/l rifampicin and 50 μl/l kanamycin. Plates were incubated for 2 daysat 28° C. and after that the number of cfu (colony forming units) wascounted. According to our estimation, 100 μl of bacterial cultures ofCryX, EHA105, GV3101 and 1CF320 contained 7.38+/−1.72, 5.00+/−1.50,2.53+/−0.87 and 6.17+/−1.37 cfu (Table 1). In parallel at 4 dpiNicotiana benthamiana leaves were scored for GFP fluorescent spots. Onaverage, 7.38+/−1.72, 5.00+/−1.50, 2.53+/−0.87 and 6.17+/−1.37fluorescent spots were produced per 100 μl of 10⁻⁷ dilution ofinfiltrated agrobacterial culture for CryX, EHA105, GV3101 and 1CF320strains, respectively. Each agrobacterial cell produced 0.46transfection loci for CryX strain and 0.01 transfection loci for allother tested strains, CryX strain being 46 times more efficient thanEHA105, GV3101 and ICF320 strains.

TABLE 1 Transfection efficiency of CryX in comparison with other strainsat concentration factor 10⁻⁷ of agrobacterial culture (pNMD560construct, 4 dpi). CryX EHA105 GV3101 ICF320 GFP spots/100 μl 2.85 +/−0.83 0.06 +/− 0.02 0.03 +/− 0.01 0.06 +/− 0.02 input culture cfu/100 μlinput 7.38 +/− 1.72 5.00 +/− 1.50 2.53 +/− 0.87 6.17 +/− 1.37 cultureGFP spots/cfu of 0.46 0.01 0.01 0.01 input culture

Example 3. Overexpression of virG Gene from LBA4404 Strain Increases theTransient Transfection Efficiency of CryX Strain

We tested the influence of overexpression of virG genes on the transienttransfection efficiency of several Agrobacterium strains. For thispurpose we created TMV-based vectors carrying the insertion of virGgenes either from GV3101 or LBA4404 strains in their plasmid backbones(FIG. 1A). First, we tested AGL1, EHA105, ICF320 and GV3101 strainsusing vectors harboring virGN54D genes from GV3101 and LBA404 strains(pNMD063 and pNMD064, respectively) as well as a vector with native virGgene sequence from LBA4404 strain (pNMD062). Nicotiana benthamianaleaves were infiltrated using syringe without needle with 10², 10³ and10⁴-fold dilutions of OD₆₀₀=1.3 of agrobacterial cultures. Photographsshowing GFP fluorescence in the uv light were taken at 4th (FIG. 3A) and6th dpi (FIG. 3B). Based on the visual evaluation, we demonstrated thestrain-specific increase of the transient transfection efficiency. As itis summarized in Table 2, overexpression of virGN54D from GV3101 strainincreased the transient transfection efficiency for GV3101 and 1CF320strains; virGN540 from LBA4404 strain improved the transienttransfection efficiency of AGA1, EHA105 and LBA4404 strains.

The CryX strain was tested similarly, as shown in FIG. 4. Nicotianabenthamiana leaves were infiltrated using syringe without needle withliquid CryX and GV3101 agrobacterial cultures of OD600=1.3 diluted 10⁻⁴and 10⁻⁵ with buffer for infiltration. GFP expression TMV-based vectorscontaining virGN54D genes from GV3101 and LBA404 strains in the plasmidbackbone (pNMD063 and pNMD064, respectively) were compared with pNMD560vector containing no virG gene insertion. FIG. 4A depicts the GFPfluorescence for the dilution 10⁻⁴ at 3 and 4 dpi FIG. 4B shows the GFPfluorescence for the dilution 10⁻⁵ at 4 and 5 dpi. We demonstrated asignificant increase of transient transfection efficiency for CryXstrain in combination with virGN54D gene from LBA4404; in contrast, theoverexpression of virGN54D gene from GV3101 strain had a negative impacton CryX-mediated transient transfection.

TABLE 2 Effect of virG overexpression on the T-DNA transfer efficiencyAgrobacterium strain virGN54D/GV3101 virGN54D/LBA4404 AGL1 no effectincrease GV3101 increase no effect ICF320 increase no effect EHA105 noeffect increase LBA4404 no effect increase

Example 4. CryX in Combinations with virGIV54D/LBA4404 ProvidesEfficient Transient Transfection of Nicotiana benthamiana in Up to10⁷-Fold Dilutions Using Leaf Infiltration

To find the maximal effective dilutions of CryX liquid cultures fortransient transfection of Nicotiana benthamiana plants, we performedsyringe infiltration of leaves with liquid CryX and GV3101 agrobacterialcultures of OD600=1.3 diluted to 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶ and 10⁻⁷ withbuffer for infiltration.

In our experiments, Agrobacterium tumefaciens strain CryX in combinationwith virGN54D from LBA4404 strain provided the most efficienttransfection of Nicotiana benthamiana plants we ever observed, resultingin the reasonable number of fluorescing spots even at the 10⁻⁷concentration factor of overnight culture (FIG. 5). It allows increasingthe dilution of agrobacterial culture used for plant infiltrationapprox. 100 to 1000-fold compared with 10³-fold dilution typically usedin Magnicon® system.

To provide a quantitative evaluation of transient transfectionefficiency for CryX strain, we estimated the ratio between the number ofcells in the bacterial suspension infiltrated in leaves and the numberof produced GFP fluorescent spots considered as a single transfectionevent. For this purpose leaves of 6-weeks old Nicotiana benthamianaplants were infiltrated using syringe without needle with 200 μl ofagrobacterial cultures of OD600=1.3 diluted 10⁻⁷ with an aqueous buffercontaining 5 mM MES, pH 5.3 and 10 mM MgCl₂. CryX agrobacterial cellscarried constructs pNMD560 (TMV-GFP vector) and pNMD062 (TMV-GFP vectorcontaining virGN54D from LBA4404 strain in the plasmid backbone). Forscoring of bacterial cells, 100 μl aliquots of bacterial suspensionsused for leaf infiltration were plated in triplicate on LB-agar platescontaining 50 μl/l rifampicin and 50 μl/l kanamycin. Plates wereincubated for 2 days at 28° C. and after that the number of cfu (colonyforming units) was counted. According to our estimation. 100 μl ofbacterial cultures contained 14.7+/−4.4 and 13.9+/−3.4 cfu for pNM0560and pNMD062 constructs, respectively (Table 3). In parallel at 5 dpiNicotiana benthamiana leaves were scored for GFP fluorescent spots. Inaverage, 16.3+/−1.5 and 24.0+/−0.0 fluorescent spots were produced per100 μl of infiltrated agrobacterial culture for pNMD560 and pNMD062constructs, respectively. In other words, each agrobacterial cellharboring the pNMD560 construct produced about 1.1 transfection loci;for the pNMD062 construct, this value was higher, 1.7, showing anincrease of the transfection efficiency due to the overexpression ofvirG gene.

Example 5, Agrobacterium Tumefaciens Strain CryX in Combination withvirGN54D/LBA4404 Shows High Spraying Transfection Efficiency for Soybean

We tested the number of Agrobacterium tumefaciens strains includingAGL1, EHA105, GV3101, ICF320, CryX and LBA4404 for the transienttransfection of soybean Glycine max L. using spraying of plants withsuspension of agrobacterial cells. For this purpose, liquid culturesharboring GUS expression vectors were grown to OD600=1.3 and diluted forspraying with buffer containing 5 mM MES pH5.3; 10 mM MgCl₂ and 0.05%(v/v) Tween®20 in the ratio 1:10. For testing of AGL1, EHA105, CryX andLBA4404 strains, we used pNMD2190 construct (35S:GUS; 35S:p19 andvirGN54D/LBA4404 in the plasmid backbone). GV3101 and ICF320 strainswere tested with pNMD2180 vector (35S:GUS; 35S:p19 and virGN54D/GV3101in the plasmid backbone). Staining of leaves for the GUS activity wasperformed at 11 days post spraying. Compared to other tested strainswhich showed no or little transfection, CryX provided significantlyhigher transient transfection rate as revealed by GUS staining (FIG.6A).

Combining surfactant and abrasive treatment, we achieved efficienttransient transfection of soybean with CryX strain for up to 10⁻²dilutions of agrobacterial cultures when GUS expression transcriptionalvector pNMD2190 was used (FIG. 6B).

Example 6. Agrobacterium Tumefaciens Strain CryX in Combination withvirGN54D/LBA4404 Shows Enhanced Transient Spraying Transfection forCotton

We tested the transient transfection of cotton with EHA105, GV3101,ICF320, and CryX strains using spraying. For this purpose liquidAgrobacterium cultures of OD₆₀₀=1.3 were diluted in the ratio 1:10 withbuffer containing 5 mM MES, pH5.3; 10 mM MgCl₂ and 0.25% (v/v) Silwet.L-77. pNMD2180 construct (35S:GUS; 35S:p19 and virGN54D/GV3101 in theplasmid backbone) was used with GV3101 and ICF320 strains, and pNMD2190(35S:GUS; 35S:p19 and virGN54D/LBA4404 in the plasmid backbone) was usedwith EHA105 and CryX strains. pNMD1971 construct (35S:GUS; 35S:p19) wasapplied as a control with all strains. GUS staining test was performedat 6 days post spraying. After the staining, few blue dots were foundfor GV3101 and ICF320 strains (data not shown). Very low transfectionefficiency was shown also for EHA105 and CryX strains used with pNMD1971construct. Compared with all other tested strains, CryX in combinationwith virGN54D from LBA4404 strain (pNM02190) demonstrated increasedtransient transfection efficiency (FIG. 7).

TABLE 3 Transfection efficiency of CryX in combination withvirGN54D/LBA4404 (pNMD062 construct) pNMD062 pNMD560 (virGN54D/ (novirG) LBA4404) Dilution of input agrobacterial culture 10 − 7 10 − 7cfu/100 μl input culture 14.7 +/− 4.4 13.9 +/− 3.4 GFP spots/100 μlinput culture, 5 dpi 16.3 +/− 1.5 24.0 +/− 0.0 GFP spots/cfu of inputculture 1.1 1.7

Example 7. ICBGE Accession of Agrobacterium tumefaciens Chry5/KYRT1Strain Shows Stronger Transient Transfection of Nicotiana benthamianaCompared to Kentucky University Accession of Same Strain

We tested two accessions of Agrobacterium tumefaciens Chry5/KYRT1 strainreceived from different laboratories, the laboratory of Dr. G. Collinsin the University of Kentucky (Lexington, USA) and the accession fromthe Institute of Cell Biology and Genetics Engineering (ICBGE, Kiev,Ukraine) for the transient transfection of Nicotiana benthamiana plants.For this purpose, plant leaves were infiltrated using needleless syringewith dilutions using concentration factors of 10⁻¹, 10⁻² and 10⁻³ of anOD₆₀₀=1.3 of agrobacterial cultures of both strain accessions harboringGFP expression TMV-based vector capable of cell-to-cell movement(TMV-GFP, pNMD560 construct) as it is shown in FIG. 8. Based on thedensity of fluorescing spots and the intensity of GFP fluorescence, weshowed higher transient transfection efficiency for ICBGE accession ifcompared with Kentucky University accession.

The content of European patent application No. 12 002 402.1 filed onApr. 3, 2012 is herein incorporated by reference in its entirety,including description, claims, figures and sequence listing.

REFERENCES

-   Akcay et al., Plant Cell Rep 28 (2009) 407-47.-   Akbulut et al, African Journal of Biotechnology 7(8) 1011-1017, 2008-   Andrews, L. B. & Curtis, W. R. (2005). Biotechnol Prog 21, 946-52.-   Barton, K. A., Binns, A, N., Matzke, A. J. & Chilton, M. D. (1983).    Cell 32, 1033-43.-   Bush A L, Pueppke S G. Appl Environ Microbial. 1991 September;    57(9):2468-72.-   Chung, M. H., Chen, M. K. & Pan, S. M, (2000). Transgenic Res 9,    471-6.-   Clough, S. J. & Bent, A. F. (1998). Plant J 16, 735-43.-   D'Aoust, M. A., Lavoie, P. O., Belles-Isles. J., Bechtold, N.,    Martel, M. & Vezina, L. P. (2009). Methods Mol Biol 483, 41-50.-   D'Aoust, M. A. Lavoie, P. O., Couture, M. M., Trepanier, S.,    Guay, J. M., Dargis, M., Mongrand, S., Landry, N., Ward, B. J. &    Vezina, L. P. (2008). Plant Biotechnol J 6, 930-40.-   De Buck, S., Jacobs, A., Van Montagu, M. & Depicker, A. (1998). Mol    Plant Microbe Interact 11, 449-57.-   De Buck, S., De Wilde, C., Van Montagu, M. & Depicker, A. (2000).    Mol Plant Microbe Interact 13, 658-65.-   de Felippes, F. F. & Weigel, D. (2010). Methods Mol Biol 592,    255-64.-   Fraley, R. T., Rogers, S. G., Horsch, R. B., Sanders, P. R.,    Flick, J. S., Adams, S. P., Bittner, M. L., Brand, L. A., Fink, C,    L., Fry, J. S., Galluppi, G. R., Goldberg, S. B., Hoffmann, N. L. &    Woo, S. C. (1983), Proc Natl Acad Sci USA 80, 4803-7.-   Gleba Y. Y. and Giritch A. (2011) Plant Viral Vectors for Protein    Expression. In Recent Advances in Plant Virology, Caister Academic    Press. ISBN 978-1-904455-75-2; pp. 387-412.-   Gleba Y. Y. and Giritch A. (2011) Vaccines, antibodies, and    pharmaceutical proteins. In Plant Biotechnology and Agriculture.    Prospects for the 21st Century. Elsevier Inc. ISBN:    978-0-12-381466-1; pp. 465-476.-   Gleba, Y., Klimyuk. V. & Marillonnet, S. (2007). Curr Opin    Biotechnol 18, 13441.-   Gleba, Y., Marillonnet, S. & Klimyuk, V. (2004). Curr Opin Plant    Biol 7, 182-8.-   Gleba, Y., Marillonnet, S. & Kiirnyuk, V. (2008). Plant virus    vectors (gene expression systems). In Encyclopedia of Virology,    Third Edition. M. H. V, van Regenmortel, Mahy. B. W. J., eds. (San    Diego, Calif.: Elsevier Academic Press), vol. 4, pp. 229-237.-   Giritch, A., Marillonnet, S., Engler, C., van Eldik, a, Botterman,    J., Klimyuk, V. & Gleba, Y. (2006). Proc Natl Acad Sci USA 103,    14701-6.-   Grant et al., Plant Cell Rep 21 (2003) 1207-1210.-   Green, B. J., Fujiki, M., Mett, V., Kaczmarczyk, J., Shamloul, M.,    Musiychuk, K., Underkoffler, S., Yusibov, V. & Mett, V. (2009).    Biotechnol J 4, 230-7.-   Huang, Z., Santi, L., LePore, K., Kilbourne, J., Arntzen, C. J. &    Mason, H. S. (2006). Vaccine 24, 2506-13.-   Jones, H. D., Doherty, A. & Sparks, C. A. (2009). Methods Mol Biol    513, 131-52.-   Ko et al, Planta 218 (2004) 536-541.-   Lee, M. W. & Yang, Y. (2006). Methods Mol Biol 323, 225-9.-   Li, X. Q., Liu, C. N., Ritchie, S. W., Peng, J. Y., Gelvin, S. B. &    Hodges, T. K. (1992). Plant Mol Biol 20, 1037-48,-   Li, J. F., Park, E., von Arnim, A. G. & Nebenfuhr, A. (2009). Plant    Methods 5, 6.-   Lindbo, J. A. (2007). TRBO: a high-efficiency tobacco mosaic virus    RNA-based overexpression vector. Plant Physiol 145, 1232-40.-   Lindbo, J. A. (2007). BMC Biotechnol 7, 52.-   Liu, C. N., Li, X. Q. & Gelvin, S. B. (1992). Plant Mol Biol 20,    1071-87.-   Lico, C., Chen, Q. & Santi, L (2008). J Cell Physiol 216, 366-77.-   Lindbo, J. A. (2007). BMC Biotechnol 7, 52.-   Marillonnet, S., Giritch, A., Gils, M., Kandzia, R., Klimyuk, V. &    Gleba, Y. (2004). Proc Natl Acad Sci USA 101, 6852-7.-   Marillonnet, S., Thoeringer, C Kandzia, R., Klimyuk, V. & Gleba, Y.    (2005). Nat Biotechnol 23, 718-23.-   Mett, V., Lyons, J., Musiychuk, K., Chichester, J. A., Brasil, T.,    Couch, R., Sherwood, R., Palmer, G. A., Streatfield, S. J. &    Yusibov, V. (2007. Vaccine 25, 3014-7.-   Palanichelvam K. et al., Mol Plant Microbe Interact, 2000 October;    13(10):1081-91.-   Plesha, M. A., Huang, T. K, Dandekar, A. M., Falk, B. W. &    McDonald, K. A. (2007). Biotechnol Prog 23, 1277-85.-   Plesha, M. A., Huang, T. K., Dandekar, A. M., Falk, B. W. &    McDonald, K. A. (2009). Biotechnol Prog 25, 722-34.-   Regnard, G. L., Halley-Stott, R. P., Tanzer, F. L., Hitzeroth, II &    Rybicki, E. P. (2010). Plant Biotechnol J 8, 38-46.-   Ryu, C. M., Anand, A., Kang, L. & Mysore, K. S. (2004). Plant J 40,    322-31.-   Santi, L, Giritch, A., Roy, C. J., Marillonnet, S., Klimyuk, V.,    Gleba, Y., Webb, R., Arntzen, C. J. & Mason, H. S. (2006). Proc Natl    Acad Sci USA 103, 861-6.-   Shang, Y., Schwinn, K. E., Bennett, M. J., Hunter, D. A., Waugh, T.    L., Pathirana, N. N., Brummell, D. A., Jameson, P. E. &    Davies, K. M. (2007). Plant Methods 3, 1.-   Shiboleth, Y. M., Arazi, T., Wang, Y. & Gal-On, A. (2001). J    Biotechnol 92, 37-46.-   Shoji, Y., Farrance, C. E., Bi, H., Shamloul, M., Green, B.,    Manceva, S., Rhee, A., Ugulava, N., Roy, G., Musiychuk, K.,    Chichester, J. A., Mett, V. & Yusibov, V. (2009). Vaccine 27,    3467-70.-   Simmons, C. W., VanderGheynst, J. S. & Upadhyaya, S. K.    (20),Biotechnol Bioeng 102, 965-70.-   Sudarshana, M. R., Plesha, M. A., Uratsu, S. L., Falk, B. W.,    Dandekar, A. M., Huang, T. K. & McDonald, K A. (2006). Plant    Biotechnol J 4, 551-9.-   Torisky et al, Plant Cell Reports 17 (1997) 102-108.-   Vaquero, C., Sack, M., Chandler, J., Drossard, J., Schuster, F.,    Monecke, M. Schillberg, S. & Fischer, R. (1999). Proc Natl Acad Sci    USA 96, 11128-33.-   Vezina, L. P., Faye, L, Lerouge, P., D'Aoust, M. A., Marquet-Blouin,    E., Burel, C., Lavoie, P. O., Bardor, M. & Gomord, V. (2009). Plant    Biotechnol J 7, 442-55.-   Voinnet, O., Rivas, S., Mestre, P. & Baulcombe, D. (2003). Plant J    33, 949-56.-   Wroblewski, T., Tomczak, A. & Michelmore, R. (2005). Plant    Biotechnol J 3, 259-73.-   Yang, Y., Li, R & Qi, M. (2000). Plant J 22, 543-51.-   Yang, L., Wang, H., Liu, J., Li, L., Fan, Y., Wang, X., Song, Y.,    Sun, S., Wang, L., Zhu, X. & Wang, X. (2008). J Biotechnol 134,    320-4.-   Zambre, M., Terryn, N., De Clercq. J., De Buck, S., Dillon, W., Van    Montagu, M., Van Der Straeten, D. & Angenon, G. (2003). Light    strongly promotes gene transfer from Agrobacterium tumefaciens to    plant cells. Planta 216, 580-6.-   Zhang, C. & Ghabrial, S. A. (2006). Virology 344, 401-11.-   Zhao, M. M., An, D. R., Zhao, J., Huang, G. H., He, Z. H. &    Chen. J. Y. (2006). Acta Biochim Biophys Sin (Shanghai) 38, 22-8.

ANNEX  SEQ ID NO: 1: Amino acid sequence from Agrobacterium LBA4404 virG protein. N54 is shown in bold.  LKHVLLVDDDVANIRHLIIEYLTIHAFKVTAVADSTQFTRVLSSATVDVVV VDLNLGREDGLEIVRNLAAKSDIPIIIISGDRLEETDKVVALELGASDA AKPFSIREFLARIRVALRURPNWRSKORRSFCRDWUNLRORRLMSEA GGEVKLTAGEFNLLLAFLEKPRDVLSREQLLIASRVRDEEVYDRSIDVLI LRLRRKLEADPSSPOLIKTARGAGYFFDADVQVSHGGTMAA  SEQ ID NO: 2: T-DNA region sequences of pNMD560, pNMD062, pNMD063 pNMD064 cctgtggttggcacatacaaatggacgaacggataaaccttttcacgcccttttaaatatccgattattctaataaacgctcttttctcttaggtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacgacaatctgatctaagctagcttggaattggtaccacgcgtttcgacaaaatttagaacgaacttaattatgatctcaaatacattgatacatatctcatctagatctaggttatcattatgtaagaaagttttgacgaatatggcacgacaaaatggctagactcgatgtaattggtatctcaactcaacattatacttataccaaacattagttagacaaaatttaaacaactattttttatgtatgcaagagtcagcatatgtataattgattcagaatcgttttgacgagttcggatgtagtagtagccattatttaatgtacatactaatcgtgaatagtgaatatgatgaaacattgtatcttattgtataaatatccataaacacatcatgaaagacactttctttcacggtctgaattaattatgatacaattctaatagaaaacgaattaaattacgttgaattgtatgaaatctaattgaacaagccaaccacgacgacgactaacgttgcctggattgactcggtttaagttaaccactaaaaaaacggagctgtcatgtaacacgcggatcgagcaggtcacagtcatgaagccatcaaagcaaaagaactaatccaagggctgagatgattaattagtttaaaaattagttaacacgagggaaaaggctgtctgacagccaggtcacgttatctttacctgtggtcgaaatgattcgtgtctgtcgattttaattatttttttgaaaggccgaaaataaagttgtaagagataaacccgcctatataaattcatatattttcctctccgctttgaagttttagttttattgcaacaacaacaacaaattacaataacaacaaacaaaatacaaacaacaacaacatggcacaatttcaacaaacaattgacatgcaaactctccaagccgctgcgggacgcaacagcttggtgaatgatttggcatctcgtcgcgtttacgataatgcagtcgaggagctgaatgctcgttccagacgtcccaaggtaataggaactttctggatctactttatttgctggatctcgatcttgttttctcaatttccttgagatctggaattcgtttaatttggatctgtgaacctccactaaatcttttggttttactagaatcgatctaagttgaccgatcagttagctcgattatagctaccagaatttggcttgaccttgatggagagatccatgttcatgttacctgggaaatgatttgtatatgtgaattgaaatctgaactgttgaagttagattgaatctgaacactgtcaatgttagattgaatctgaacactgtttaaggttagatgaagtttgtgtatagattcttcgaaactttaggatttgtagtgtcgtacgttgaacagaaagctatttctgattcaatcagggtttatttgactgtattgaactctttttgtgtgtttgcaggtccacttctccaaggcagtgtctacggaacagaccctgattgcaacaaacgcatatccggagttcgagatttcctttactcatacgcaatccgctgtgcactccttggccggaggccttcggtcacttgagttggagtatctcatgatgcaagttccgttcggttctctgacgtacgacatcggcggtaacttttccgcgcaccttttcaaagggcgcgattacgttcactgctgcatgcctaatctggatgtacgtgacattgctcgccatgaaggacacaaggaagctatttacagttatgtgaatcgtttgaaaaggcagcagcgtcctgtgcctgaataccagagggcagctttcaacaactacgctgagaacccgcacttcgtccattgcgacaaacctttccaacagtgtgaattgacgacagcgtatggcactgacacctacgctgtagctctccatagcatttatgatatccctgttgaggagttcggttctgcgctactcaggaagaatgtgaaaacttgtttcgcggcctttcatttccatgagaatatgcttctagattgtgatacagtcacactcgatgagattggagctacgttccagaaatcaggtaacattccttagttacctttcttttctttttccatcataagtttatagattgtacatgctttgagatttttctttgcaaacaatctcaggtgataacctgagcttcttcttccataatgagagcactctcaattacacccacagcttcagcaacatcatcaagtacgtgtgcaagacgttcttccctgctagtcaacgcttcgtgtaccacaaggagttcctggtcactagagtcaacacttggtactgcaagttcacgagagtggatacgttcactctgttccgtggtgtgtaccacaacaatgtggattgcgaagagttttacaaggctatggacgatgcgtggcactacaaaaagacgttagcaatgcttaatgccgagaggaccatcttcaaggataacgctgcgttaaacttctggttcccgaaggtgctcttgaaattggaagtcttcttttgttgtctaaacctatcaatttctttgcggaaatttatttgaagctgtagagttaaaattgagtcttttaaacttttgtaggtgagagacatggttatcgtccctctcttgacgcttctatcacaactggtaggatgtctaggagagaggttatggtgaacaaggacttcgtctacacggtcctaaatcacatcaagacctatcaagctaaggcactgacgtacgcaaacgtgctgagcttcgtggagtctattaggtctagagtgataattaacggtgtcactgccaggtaagttgttacttatgattgttttcctctctgctacatgtattttgttgttcatttctgtaagatataagaattgagttttcctctgatgatattattaggtctgaatgggacacagacaaggcaattctaggtccattagcaatgacattcttcctgatcacgaagctgggtcatgtgcaagatgaaataatcctgaaaaagttccagaagttcgacagaaccaccaatgagctgatttggacaagtctctgcgatgccctgatgggggttattccctcggtcaaggagacgcttgtgcgcggtggttttgtgaaagtagcagaacaagccttagagatcaaggttagtatcatatgaagaaatacctagtttcagttgatgaatgctattttctgacctcagttgttctcttttgagaattatttctttctaatttgcctgatttttctattaattcattaggttcccgagctatactgtaccttcgccgaccgattggtactacagtacaagaaggcggaggagttccaatcgtgtgatctttccaaacctctagaagagtcagagaagtactacaacgcattatccgagctatcagtgcttgagaatctcgactcttttgacttagaggcgtttaagactttatgtcagcagaagaatgtggacccggatatggcagcaaaggtaaatcctggtccacacttttacgataaaaacacaagattttaaactatgaactgatcaataatcattcctaaaagaccacacttttgttttgtttctaaagtaatttttactgttataacaggtggtcgtagcaatcatgaagtcagaattgacgttgcctttcaagaaacctacagaagaggaaatctcggagtcgctaaaaccaggagaggggtcgtgtgcagagcataaggaagtgttgagcttacaaaatgatgctccgttcccgtgtgtgaaaaatctagttgaaggttccgtgccggcgtatggaatgtgtcctaagggtggtggtttcgacaaattggatgtggacattgctgatttccatctcaagagtgtagatgcagttaaaaagggaactatgatgtctgcggtgtacacagggtctatcaaagttcaacaaatgaagaactacatagattacttaagtgcgtcgctggcagctacagtctcaaacctctgcaaggtaagaggtcaaaaggtttccgcaatgatccctctttttttgtttctctagtttcaagaatttgggtatatgactaacttctgagtgttccttgatgcatatttgtgatgagacaaatgtttgttctatgttttaggtgcttagagatgttcacggcgttgacccagagtcacaggagaaatctggagtgtgggatgttaggagaggacgttggttacttaaacctaatgcgaaaagtcacgcgtggggtgtggcagaagacgccaaccacaagttggttattgtgttactcaactgggatgacggaaagccggtttgtgatgagacatggttcagggtggcggtgtcaagcgattccttgatatattcggatatgggaaaacttaagacgctcacgtcttgcagtccaaatggtgagccaccggagcctaacgccaaagtaattttggtcgatggtgttcccggttgtggaaaaacgaaggagattatcgaaaaggtaagttctgcatttggttatgctccttgcattttaggtgttcgtcgctcttccatttccatgaatagctaagattttttttctctgcattcattcttcttgcctcagttctaactgtttgtggtatttttgttttaattattgctacaggtaaacttctctgaagacttgattttagtccctgggaaggaagcttctaagatgatcatccggagggccaaccaagctggtgtgataagagcggataaggacaatgttagaacggtggattccttcttgatgcatccttctagaagggtgtttaagaggttgtttatcgatgaaggactaatgctgcatacaggttgtgtaaatttcctactgctgctatctcaatgtgacgtcgcatatgtgtatggggacacaaagcaaattccgttcatttgcagagtcgcgaactttccgtatccagcgcattttgcaaaactcgtcgctgatgagaaggaagtcagaagagttacgctcaggtaaagcaactgtgttttaatcaatttcttgtcaggatatatggattataacttaatttttgagaaatctgtagtatttggcgtgaaatgagtttgctttttggtttctcccgtgttataggtgcccggctgatgttacgtatttccttaacaagaagtatgacggggcggtgatgtgtaccagcgcggtagagagatccgtgaaggcagaagtggtgagaggaaagggtgcattgaacccaataaccttaccgttggagggtaaaattttgaccttcacacaagctgacaagttcgagttactggagaagggttacaaggtaaagtttccaactttcctttaccatatcaaactaaagttcgaaactttttatttgatcaacttcaaggccacccgatctttctattcctgattaatttgtgatgaatccatattgacttttgatggttacgcaggatgtgaacactgtgcacgaggtgcaaggggagacgtacgagaagactgctattgtgcgcttgacatcaactccgttagagatcatatcgagtgcgtcacctcatgttttggtggcgctgacaagacacacaacgtgttgtaaatattacaccgttgtgttggacccgatggtgaatgtgatttcagaaatggagaagttgtccaatttccttcttgacatgtatagagttgaagcaggtctgtctttcctatttcatatgtttaatcctaggaatttgatcaattgattgtatgtatgtcgatcccaagactttcttgttcacttatatcttaactctctcttgctgtttcttgcaggtgtccaatagcaattacaaatcgatgcagtattcaggggacagaacttgtttgttcagacgcccaagtcaggagattggcgagatatgcaattttactatgacgctcttcttcccggaaacagtactattctcaatgaatttgatgctgttacgatgaatttgagggatatttccttaaacgtcaaagattgcagaatcgacttctccaaatccgtgcaacttcctaaagaacaacctattttcctcaagcctaaaataagaactgcggcagaaatgccgagaactgcaggtaaaatattggatgccagacgatattctttctttgatttgtaactttttcctgtcaaggtcgataaattttattttttttggtaaaaggtcgataatttttttttggagccattatgtaattttcctaattaactgaaccaaaattatacaaaccaggtttgctggaaaatttggttgcaatgatcaaaagaaacatgaatgcgccggatttgacagggacaattgacattgaggatactgcatctctggtggttgaaaagttttgggattcgtatgttgacaaggaatttagtggaacgaacgaaatgaccatgacaagggagagcttctccaggtaaggacttctcatgaatattagtggcagattagtgttgttaaagtctttggttagataatcgatgcctcctaattgtccatgttttactggttttctacaattaaaggtggctttcgaaacaagagtcatctacagttggtcagttagcggactttaactttgtggatttgccggcagtagatgagtacaagcatatgatcaagagtcaaccaaagcaaaagttagacttgagtattcaagacgaatatcctgcattgcagacgatagtctaccattcgaaaaagatcaatgcgattttcggtccaatgttttcagaacttacgaggatgttactcgaaaggattgactcttcgaagtttctgttctacaccagaaagacacctgcacaaatagaggacttctttctgacctagactcaacccaggcgatggaaattctggaactcgacatttcgaagtacgataagtcacaaaacgagttccattgtgctgtagagtacaagatctgggaaaagttaggaattgatgagtggctagctgaggtctggaaacaaggtgagttcctaagttccatttttttgtaatccttcaatgttattttaacttttcagatcaacatcaaaattaggttcaattttcatcaaccaaataatatttttcatgtatatataggtcacagaaaaacgaccttgaaagattatacggccggaatcaaaacatgtctttggtatcaaaggaaaagtggtgatgtgacaacctttattggtaataccatcatcattgccgcatgtttgagctcaatgatccccatggacaaagtgataaaggcagctttttgtggagacgatagcctgatttacattcctaaaggtttagacttgcctgatattcaggcgggcgcgaacctcatgtggaacttcgaggccaaactcttcaggaagaagtatggttacttctgtggtcgttatgttattcaccatgatagaggagccattgtgtattacgatccgcttaaactaatatctaagttaggttgtaaacatattagagatgttgttcacttagaagagttacgcgagtctttgtgtgatgtagctagtaacttaaataattgtgcgtattttcacagttagatgaggccgttgccgaggttcataagaccgcggtaggcggttcgtttgctttttgtagtataattaagtatttgtcagataagagattgtttagagatttgttctttgtttgataatgtcgatagtctcgtacgaacctaaggtgagtgatttcctcaatctttcgaagaaggaagagatcttgccgaaggctctaacgaggttaaaaaccgtgtctattagtactaaagatattatatctgtcaaggagtcggagactttgtgtgatatagatttgttaatcaatgtgccattagataagtatagatatgtgggtatcctaggagccgtttttaccggagagtggctagtgccagacttcgttaaaggtggagtgacgataagtgtgatagataagcgtctggtgaactcaaaggagtgcgtgattggtacgtacagagccgcagccaagagtaagaggttccagttcaaattggttccaaattactttgtgtccaccgtggacgcaaagaggaagccgtggcaggtaaggatttttatgatatagtatgcttatgtattttgtactgaaagcatatcctgcttcattgggatattactgaaagcatttaactacatgtaaactcacttgatgatcaataaacttgattttgcaggttcatgttcgtatacaagacttgaagattgaggcgggttggcagccgttagctctggaagtagtttcagttgctatggtcaccaataacgttgtcatgaagggtttgagggaaaaggtcgtcgcaataaatgatccggacgtcgaaggtttcgaaggtaagccatcttcctgcttatttttataatgaacatagaaataggaagttgtgcagagaaactaattaacctgactcaaaatctaccctcataattgttgtttgatattggtcttgtattttgcaggtgtggttgacgaattcgtcgattcggttgcagcatttaaagcggttgacaactttaaaagaaggaaaaagaaggttgaagaaaagggtgtagtaagtaagtataagtacagaccggagaagtacgccggtcctgattcgtttaatttgaaagaagaaaacgtcttacaacattacaaacccgaataatcgataactcgagtatttttacaacaattaccaacaacaacaaacaacaaacaacattacaattacatttacaattatcatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcagctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactcacggcatggacgagctgtacaagtaaagcggcccctagagcgtggtgcgcacgatagcgcatagtgtttttctctccacttgaatcgaagagatagacttacggtgtaaatccgtaggggtggcgtaaaccaaattacgcaatgttttgggttccatttaaatcgaaaccccttatttcctggatcacctgttaacgcacgtttgacgtgtattacagtgggaataagtaaaagtgagaggttcgaatcctccctaaccccgggtaggggcccagcggccgctctagctagagtcaagcagatcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgacctgcatccaccccagtacattaaaaacgtccgcaatgtgttattaagttgtcataagcgtcaatttgfttacacc matatatcctgccaccagccaccac ccc ac gcagctcggcac atcaccactcgatacaggcagc ccatcac  SEQ ID NO: 3:  Sequence of T-DNA region of pNMD570 cctgtggttggcacatacaaatggacgaacggataaaccttttcacgcccttttaaatatccgattattctaataaacgctcttttctcttaggtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacgacaatctgatctaagctagcttggaattggtaccacgcgtttcgacaaaatttagaacgaacttaattatgatctcaaatacattgatacatatctcatctagatctaggttatcattatgtaagaaagttttgacgaatatggcacgacaaaatggctagactcgatgtaattggtatctcaactcaacattatacttataccaaacattagttagacaaaatttaaacaactattttttatgtatgcaagagtcagcatatgtataattgattcagaatcgttttgacgagttcggatgtagtagtagccattatttaatgtacatactaatcgtgaatagtgaatatgatgaaacattgtatcttattgtataaatatccataaacacatcatgaaagacactttctttcacggtctgaattaattatgatacaattctaatagaaaacgaattaaattacgttgaattgtatgaaatctaattgaacaagccaaccacgacgacgactaacgttgcctggattgactcggtttaagttaaccactaaaaaaacggagctgtcatgtaacacgcggatcgagcaggtcacagtcatgaagccatcaaagcaaaagaactaatccaagggctgagatgattaattagtttaaaaattagttaacacgagggaaaaggctgtctgacagccaggtcacgttatctttacctgtggtcgaaatgattcgtgtctgtcgattttaattatttttttgaaaggccgaaaataaagttgtaagagataaacccgcctatataaattcatatattttcctctccgctttgaagttttagttttattgcaacaacaacaacaaattacaataacaacaaacaaaatacaaacaacaacaacatggcacaatttcaacaaacaattgacatgcaaactctccaagccgctgcgggacgcaacagcttggtgaatgatttggcatctcgtcgcgtttacgataatgcagtcgaggagctgaatgctcgttccagacgtcccaaggtaataggaactttctggatctactttatttgctggatctcgatcttgttttctcaatttccttgagatctggaattcgtttaatttggatctgtgaacctccactaaatcttttggttttactagaatcgatctaagttgaccgatcagttagctcgattatagctaccagaatttggcttgaccttgatggagagatccatgttcatgttacctgggaaatgatttgtatatgtgaattgaaatctgaactgttgaagttagattgaatctgaacactgtcaatgttagattgaatctgaacactgtttaaggttagatgaagtttgtgtatagattcttcgaaactttaggatttgtagtgtcgtacgttgaacagaaagctatttctgattcaatcagggtttatttgactgtattgaactctttttgtgtgtttgcaggtccacttctccaaggcagtgtctacggaacagaccctgattgcaacaaacgcatatccggagttcgagatttcctttactcatacgcaatccgctgtgcactccttggccggaggccttcggtcacttgagttggagtatctcatgatgcaagttccgttcggttctctgacgtacgacatcggcggtaacttttccgcgcaccttttcaaagggcgcgattacgttcactgctgcatgcctaatctggatgtacgtgacattgctcgccatgaaggacacaaggaagctatttacagttatgtgaatcgtttgaaaaggcagcagcgtcctgtgcctgaataccagagggcagctttcaacaactacgctgagaacccgcacttcgtccattgcgacaaacctttccaacagtgtgaattgacgacagcgtatggcactgacacctacgctgtagctctccatagcatttatgatatccctgttgaggagttcggttctgcgctactcaggaagaatgtgaaaacttgtttcgcggcctttcatttccatgagaatatgcttctagattgtgatacagtcacactcgatgagattggagctacgttccagaaatcaggtaacattccttagttacctttcttttctttttccatcataagtttatagattgtacatgctttgagatttttctttgcaaacaatctcaggtgataacctgagcttcttcttccataatgagagcactctcaattacacccacagcttcagcaacatcatcaagtacgtgtgcaagacgttcttccctgctagtcaacgcttcgtgtaccacaaggagttcctggtcactagagtcaacacttggtactgcaagttcacgagagtggatacgttcactctgttccgtggtgtgtaccacaacaatgtggattgcgaagagttttacaaggctatggacgatgcgtggcactacaaaaagacgttagcaatgcttaatgccgagaggaccatcttcaaggataacgctgcgttaaacttctggttcccgaaggtgctcttgaaattggaagtcttcttttgttgtctaaacctatcaatttctttgcggaaatttatttgaagctgtagagttaaaattgagtcttttaaacttttgtaggtgagagacatggttatcgtccctctctttgacgcttctatcacaactggtaggatgtctaggagagaggttatggtgaacaaggacttcgtctacacggtcctaaatcacatcaagacctatcaagctaaggcactgacgtacgcaaacgtgctgagcttcgtggagtctattaggtctagagtgataattaacggtgtcactgccaggtaagttgttacttatgattgttttcctctctgctacatgtattttgttgttcatttctgtaagatataagaattgagttttcctctgatgatattattaggtctgaatgggacacagacaaggcaattctaggtccattagcaatgacattcttctgatcacgaagctgggtcatgtgcaagatgaaataatcctgaaaagttccgaagttcgacagaaccaccaatgagctgatttggacaagtctctgcgatgccctgatgggggttattccctcggtcaaggagacgcttgtgcgcggtggttttgtgaaagtagcagaacaagccttagagatcaaggttagtatcatatgaagaaatacctagtttcagttgatgaatgctattttctgacctcagttgttctcttttgagaattatttcttttctaatttgcctgatttttctattaattcattaggttcccgagctatactgtaccttcgccgaccgattggtactacagtacaagaaggcggaggagttccaatcgtgtgatctttccaaacctctagaagagtcagagaagtactacaacgcattatccgagctatcagtgcttgagaatctcgactcttttgattagaggcgtttaagactttatgtcagcagaagaatgtggacccggatatggcagcaaaggtaaatcctggtccacacttttacgataaaaacacaagattttaaactatgaactgatcaataatcattcctaaaagaccacacacttttgttttgtttctaaagtaatttttactgttataacaggtggtcgtagcaatcatgaagtcagaattgacgttgcctttcaagaaacctacagaagaggaaatctcggagtcgctaaaaccaggagaggggtcgtgtgcagagcataaggaagtgttgagcttacaaaatgatgctccgttcccgtgtgtgaaaaatctagttgaaggttccgtgccggcgtatggaatgtgtcctaagggtggtggtttcgacaaattggatgtggacattgctgatttccatctcaagagtgtagatgcagttaaaaagggaactatgatgtctgcggtgtacacagggtctatcaaagttcaacaaatgaagaactacatagattacttaagtgcgtcgctggcagctacagtctcaaacctctgcaaggtaagaggtcaaaaggtttccgcaatgatccctctttttttgtttctctagtttcaagaatttgggtatatgactaacttctgagtgttccttgatgcatatttgtgatgagacaaatgtttgttctatgttttaggtgcttagagatgttcacggcgttgacccagagtcacaggagaaatctggagtgtgggatgttaggagaggacgttggttacttaaacctaatgcgaaaagtcacgcgtggggtgtggcagaagacgccaaccacaagttggttattgtgttactcaactgggatgacggaaagccggtttgtgatgagacatggttcagggtggcggtgtcaagcgattccttgatatattcggatatgggaaaacttaagacgctcacgtcttgcagtccaaatggtgagccaccggagcctaacgccaaagtaattttggtcgatggtgttcccggttgtggaaaaacgaaggagattatcgaaaaggtaagttctgcatttggttatgctccttgcattttaggtgttcgtcgctcttccatttccatgaatagctaagattttttttctctgcattcattcttcttgcctcagttctaactgtttgtggtatttttgttttaattattgctacaggtaaacttctctgaagacttgattttagtccctgggaaggaagcttctaagatgatcatccggagggccaaccaagctggtgtgataagagcggataaggacaatgttagaacggtggattccttcttgatgcatccttctagaagggtgtttaagaggttgtttatcgatgaaggactaatgctgcatacaggttgtgtaaatttcctactgctgctatctcaatgtgacgtcgcatatgtgtatggggacacaaagcaaattccgttcatttgcagagtcgcgaactttccgtatccagcgcattttgcaaaactcgtcgctgatgagaaggaagtcagaagagttacgctcaggtaaagcaactgtgttttaatcaatttcttgtcaggatatatggattataacttaatttttgagaaatctgtagtatttggcgtgaaatgagtttgctttttggtttctcccgtgttataggtgcccggctgatgttacgtatttccttaacaagaagtatgacggggcggtgatgtgtaccagcgcggtagagagatccgtgaaggcagaagtggtgagaggaaagggtgcattgaacccaataaccttaccgttggagggtaaaattttgaccttcacacaagctgacaagttcgagttactggagaagggttacaaggtaaagtttccaactttcctttaccatatcaaactaaagttcgaaactttttatttgatcaacttcaaggccacccgatctttctattcctgattaatttgtgatgaatccatattgacttttgatggttacgcaggatgtgaacactgtgcacgaggtgcaaggggagacgtacgagaagactgctattgtgcgcttgacatcaactccgttagagatcatatcgagtgcgtcacctcatgttttggtggcgctgacaagacacacaacgtgttgtaaatattacaccgttgtgttggacccgatggtgaatgtgatttcagaaatggagaagttgtccaatttccttcttgacatgtatagagttgaagcaggtctgtctttcctatttcatatgtttaatcctaggaatttgatcaattgattgtatgtatgtcgatcccaagactttcttgttcacttatatcttaactctctctttgctgtttcttgcaggtgtccaatagcaattacaaatcgatgcagtattcaggggacagaacttgtttgttcagacgcccaagtcaggagattggcgagatatgcaattttactatgacgctcttcttcccggaaacagtactattctcaatgaatttgatgctgttacgatgaatttgagggatatttccttaaacgtcaaagattgcagaatcgacttctccaaatccgtgcaacttcctaaagaacaacctattttcctcaagcctaaaataagaactgcggcagaaatgccgagaactgcaggtaaaatattggatgccagacgatattctttcttttgatttgtaactttttcctgtcaaggtcgataaattttattttttttggtaaaaggtcgataatttttttttggagccattatgtaattttcctaattaactgaaccaaaattatacaaaccaggtttgctggaaaatttggttgcaatgatcaaaagaaacatgaatgcgccggatttgacagggacaattgacattgaggatactgcatctctggtggttgaaaagttttgggattcgtatgttgacaaggaatttagtggaacgaacgaaatgaccatgacaagggagagcttctccaggtaaggacttctcatgaatattagtggcagattagtgttgttaaagtctttggttagataatcgatgcctcctaattgtccatgttttactggttttctacaattaaaggtggctttcgaaacaagagtcatctacagttggtcagttagcggactttaactttgtggatttgccggcagtagatgagtacaagcatatgatcaagagtcaaccaaagcaaaagttagacttgagtattcaagacgaatatcctgcattgcagacgatagtctaccattcgaaaaagatcaatgcgattttcggtccaatgttttcagaacttacgaggatgttactcgaaaggattgactcttcgaagtttctgttctacaccagaaagacacctgcacaaatagaggacttcttttctgacctagactcaacccaggcgatggaaattctggaactcgacatttcgaagtacgataagtcacaaaacgagttccattgtgctgtagagtacaagatctgggaaaagttaggaattgatgagtggctagctgaggtctggaaacaaggtgagttcctaagttccatttttttgtaatccttcaatgttattttaacttttcagatcaacatcaaaattaggttcaattttcatcaaccaaataatatttttcatgtatatataggtcacagaaaaacgaccttgaaagattatacggccggaatcaaaacatgtctttggtatcaaaggaaaagtggtgatgtgacaacctttattggtaataccatcatcattgccgcatgtttgagctcaatgatccccatggacaaagtgataaaggcagctttttgtggagacgatagcctgatttacattcctaaaggtttagacttgcctgatattcaggcgggcgcgaacctcatgtggaacttcgaggccaaactcttcaggaagaagtatggttacttctgtggtcgttatgttattcaccatgatagaggagccattgtgtattacgatccgcttaaactaatatctaagttaggttgtaaacatattagagatgttgttcacttagaagagttacgcgagtctttgtgtgatgtagctagtaacttaaataattgtgcgtatttttcacagttagatgaggccgttgccgaggttcataagaccgcggtaggcggttcgtttgctttttgtagtataattaagtatttgtcagataagagattgtttagagattgttctttgtttgataatgtcgatagtctcgtacgaacctaaggtgagtgatttcctcaatctttcgaagaaggaagagatcttgccgaaggctctaacgaggttaaaaaccgtgtctattagtactaaagatattatatctgtcaaggagtcggagactttgtgtgatatagatttgttaatcaatgtgccattagataagtatagatatgtgggtatcctagctaggagccgtttttaccggagagtggctagtgccagacttcgttaaaggtggagtgacgataagtgtgatagataagcgtctggtgaactcaaaggagtgcgtgattggtacgtacagagccgcagccaagagtaagaggttccagttcaaattggttccaaattactttgtgtccaccgtggacgcaaagaggaagccgtggcaggtaaggatttttatgatatagtatgcttatgtattttgtactgaaagcatatcctgcttcattgggatattactgaaagcatttaactacatgtaaactcacttgatgatcaataaacttgattttgcaggttcatgttcgtatacaagacttgaagattgaggcgggttggcagccgttagctctggaagtagtttcagttgctatggtcaccaataacgttgtcatgaagggtttgagggaaaaggtcgtcgcaataaatgatccggacgtcgaaggtttcgaaggtaagccatcttcctgcttatttttataatgaacatagaaataggaagttgtgcagagaaactaattaacctgactcaaaatctaccctcataattgttgtttgatattggtcttgtattttgcaggtgtggttgacgaattcgtcgattcggttgcagcatttaaagcggttgacaactttaaaagaaggaaaaagaaggttgaagaaaagggtgtagtaagtaagtataagtacagaccggagaagtacgccggtcctgattcgtttaatttgaaagaagaaaacgtcttacaacattacaaacccgaataatcgataactcgagtatttttacaacaattaccaacaacaacaaacaacaaacaacattacaattacatttacaattatcatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccttcagctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactcacggcatggacgagctgtacaagtaaagcggcccctagagcgtggtgcgcacgatagcgcatagtgtttttctctccacttgaatcgaagagatagacttacggtgtaaatccgtaggggtggcgtaaaccaaaattacgcaatgttttgggttccatttaaatcgaaaccccttatttcctggatcacctgttaacgcacgtttgacgtgtattacagtgggaataagtaaaagtgagaggttcgaatcctccctaaccccgggtaggggcccagcggccgctctagctagagtcaagcagatcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgacctgcatccaccccagtacattaaaaacgtccgcaatgtgttattaagttgtctaagcgtcaatttgtttacaccacaatatatcctgccaccagccagccaacagctccccgaccggcagctcggcacaaaatcaccactcgatacaggcagcccatcag SEQ ID NO: 4:  Sequence of T-DNA region of pNMD620 cctgtggttggcacatacaaatggacgaacggataaaccttttcacgcccttttaaatatccgattattctaataaacgctcttttctcttaggtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacgacaatctgatctaagctaggcatgcctgcaggtcaacatggtggagcacgacacgcttgtctactccaaaaatatcaaagatacagtctcagaagaccaaagggcaattgagacttttcaacaaagggtaatatccggaaacctcctcggattccattgcccagctatctgtcactttattgtgaagatagtggaaaaggaaggtggctcctacaaatgccatcattgcgataaaggaaacgccatcgttgaagatgcctctgccgacagtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgatatctccactgacgtaagggatgacgcacaatcccactatccttcgcaagacccttcctctatataaggaagttcatttcatttggagaggagaaaactaaaccatacaccaccaacacaaccaaacccaccacgcccaattgttacacacccgcttgaaaaagaaagtttaacaaatggccaaggtgcgcgaggtttaccaatcttttacagactccaccacaaaaactctccatccaagatgaggcttatagaaacattcgccccatcatggaaaaacacaaactagctaacccttacgctcaaacggttgaagcggctaatgatctagaggggttcggcatagccaccaatccctatagcattgaattgcatacacatgcagccgctaagaccatagagaataaacttctagaggtgcttggttccatcctaccacaagaacctgttacatttatgtttcttaaacccagaaagctaaactacatgagaagaaacccgcggatcaaggacattttccaaaatgttgccattgaaccaagagacgtagccaggtaccccaaggaaacaataattgacaaactcacagagatcacaacggaaacagcatacattagtgacactctgcacttcttggatccgagctacatagtggagacattccaaaactgcccaaaattgcaaacattgtatgcgaccttagttctccccgttgaggcagcctttaaaatggaaagcactcacccgaacatatacagcctcaaatacttcggagatggtttccagtatataccaggcaaccatggtggcggggcataccatcatgaattcgctcatctacaatggctcaaagtgggaaagatcaagtggagggaccccaaggatagctttctcggacatctcaattacacgactgagcaggttgagatgcacacagtgacagtacagttgcaggaatcgttcgcggcaaaccacttgtactgcatcaggagaggagacttgctcacaccggaggtgcgcactttcggccaacctgacaggtacgtgattccaccacagatcttcctcccaaaagttcacaactgcaagaagccgattctcaagaaaactatgatgcagctcttcttgtatgttaggacagtcaaggtcgcaaaaaattgtgacatttttgccaaagtcagacaattaattaaatcatctgacttggacaaatactctgctgtggaactggtttacttagtaagctacatggagttccttgccgatttacaagctaccacctgcttctcagacacactttctggtggcttgctaacaaagacccttgcaccggtgagggcttggatacaagagaaaaagatgcagctgtttggtcttgaggactacgcgaagttagtcaaagcagttgatttccacccggtggatttttctttcaaagtggaaacttgggacttcagattccaccccttgcaagcgtggaaagccttccgaccaagggaagtgtcggatgtagaggaaatggaaagtttgttctcagatggggacctgcttgattgcttcacaagaatgccagcttatgcggtaaacgcagaggaagatttagctgcaatcaggaaaacgcccgagatggatgtcggtcaagaagttaaagagcctgcaggagacagaaatcaatactcaaaccctgcagaaactttcctcaacaagctccacaggaaacacagtagggaggtgaaacaccaggccgcaaagaaagctaaacgcctagctgaaatccaggagtcaatgagagctgaaggtgatgccgaaccaaatgaaataagcgggacgatgggggcaatacccagcaacgccgaacttcctggcacgaatgatgccagacaagaactcacactcccaaccactaaacctgtccctgcaaggtgggaagatgcttcattcacagattctagtgtggaagaggagcaggttaaactccttggaaaagaaaccgttgaaacagcgacgcaacaagtcatcgaaggacttccttggaaacactggattcctcaattaaatgctgttggattcaaggcgctggaaattcagagggataggagtggaacaatgatcatgcccatcacagaaatggtctccgggctggaaaaagaggacttccctgaaggaactccaaaagagttggcacgagaattgttcgctatgaacagaagccctgccaccatccctttggacctgcttagagccagagactacggcagtgatgtaaagaacaagagaattggtgccatcacaaagacacaggcaacgagttggggcgaatacttgacaggaaagatagaaagcttaactgagaggaaagttgcgacttgtgtcattcatggagctggaggttctggaaaaagtcatgccatccagaaggcattgagagaaattggcaagggctcggacatcactgtagtcctgccgaccaatgaactgcggctagattggagtaagaaagtgcctaacactgagccctatatgttcaagacctctgaaaaggcgttaattgggggaacaggcagcatagtcatctttgacgattactcaaaacttcctcccggttacatagaagccttagtctgtttctactctaaaatcaagctaatcattctaacaggagatagcagacaaagcgtctaccatgaaactgctgaggacgcctccatcaggcatttgggaccagcaacagagtacttctcaaaatactgccgatactatctcaatgccacacaccgcaacaagaaagatcttgcgaacatgcttggtgtctacagtgagagaacgggagtcaccgaaatcagcatgagcgccgagttcttagaaggaatcccaactttggtaccctcggatgagaagagaaagctgtacatgggcaccgggaggaatgacacgttcacatacgctggatgccaggggctaactaagccgaaggtacaaatagtgttggaccacaacacccaagtgtgtagcgcgaatgtgatgtacacggcactttctagagccaccgataggattcacttcgtgaacacaagtgcaaattcctctgccttctgggaaaagttggacagcaccccttacctcaagactttcctatcagtggtgagagaacaagcactcagggagtacgagccggcagaggcagagccaattcaagagcctgagccccagacacacatgtgtgtcgagaatgaggagtccgtgctagaagagtacaaagaggaactcttggaaaagtttgacagagagatccactctgaatcccatggtcattcaaactgtgtccaaactgaagacacaaccattcagttgttttcgcatcaacaagcaaaagatgagaccctcctctgggcgactatagatgcgcggctcaagaccagcaatcaagaaacaaacttccgagaattcctgagcaagaaggacattggggacgttctgtttttaaactaccaaaaagctatgggtttacccaaagagcgtattcctttttcccaagaggtctgggaagcttgtgcccacgaagtacaaagcaagtacctcagcaagtcaaagtgcaacttgatcaatgggactgtgagacagagcccagacttcgatgaaaataagattatggtattcctcaagtcgcagtgggtcacaaaggtggaaaaactaggtctacccaagattaagccaggtcaaaccatagcagccttttaccagcagactgtgatgctttttggaactatggctaggtacatgcgatggttcagacaggctttccagccaaaagaagtcttcataaactgtgagaccacgccagatgacatgtctgcatgggccttgaacaactggaatttcagcagacctagcttggctaatgactacacagctttcgaccagtctcaggatggagccatgttgcaatttgaggtgctcaaagccaaacaccactgcataccagaggaaatcattcaggcatacatagatattaagactaatgcacagattttcctaggcacgttatcaattatgcgcctgactggtgaaggtcccacttttgatgcaaacactgagtgcaacatagcttacacccatacaaagtttgacatcccagccggaactgctcaagtttatgcaggagacgactccgcactggactgtgttccagaagtgaagcatagtttccacaggcttgaggacaaattactcctaaagtcaaagcctgtaatcacgcagcaaaagaagggcagttggcctgagttttgtggttggctgatcacaccaaaaggggtgatgaaagacccaattaagctccatgttagcttaaaattggctgaagctaagggtgaactcaagaaatgtcaagattcctatgaaattgatctgagttatgcctatgaccacaaggactctctgcatgacttgttcgatgagaaacagtgtcaggcacacacactcacttgcagaacactaatcaagtcagggagaggcactgtctcactttcccgcctcagaaactttctttcaaccgttaagttaccttgagatttgaataagatggatattctcatcagtagtttgaaaagtttaggttattctaggacttccaaatctttagattcaggacctttggtagtacatgcagtagccggagccggtaagtccacagccctaaggaagttgatcctcagacacccaacattcaccgtgcatacactcggtgtccctgacaaggtgagtatcagaactagaggcatacagaagccaggacctattcctgagggcaacttcgcaatcctcgatgagtatactttggacaacaccacaaggaactcataccaggcactttttgctgacccttatcaggcaccggagtttagcctagagccccacttctacttggaaacatcatttcgagttccgaggaaagtggcagatttgatagctggctgtggcttcgatttcgagacgaactcaccggaagaagggcacttagagatcactggcatattcaaagggcccctactcggaaaggtgatagccattgatgaggagtctgagacaacactgtccaggcatggtgttgagtttgttaagccctgccaagtgacgggacttgagttcaaagtagtcactattgtgtctgccgcaccaatagaggaaattggccagtccacagctttctacaacgctatcaccaggtcaaagggattgacatatgtccgcgcagggccataggctgaccgctccggtcaattctgaaaagtgtacatagtattaggtctatcatttgctttagtttcaattacctttctgctttctagaaatagcttaccccacgtcggtgacaacattcacagcttgccacacggaggagcttacagagacggcaccaaagcaatcttgtacaactccccaaatctagggtcacgagtgagtctacacaacggaaagaacgcagcatttgctgccgttttgctactgactttgctgatctatggaagtaaatacatatctcaacgcaatcatacttgtgcttgtggtaacaatcatagcagtcattagcacttccttagtgaggactgaaccttgtgtcatcaagattactggggaatcaatcacagtgttggcttgcaaactagatgcagaaaccataagggccattgccgatctcaagccactctccgttgaacggttaagtttccattgatactcgaaagaggtcagcaccagctagcaacaaacaagaaaggtatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctcgcaccaccggaagctgcccgtgccctggcccaccctcgtgaccaccttcagctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactcacggcatggacgagctgtacaagtaagcttggtcgtatcactggaacaacaaccgctgaggctgttgtcactctaccaccaccataactacgtctacataaccgacgcctaccccagtttcatagtattttctggtttgattgtatgaataatataaataaaaaaaaaaaaaaaaaaaaaaaactagtgagctcttctgtcagcgggcccactgcatccaccccagtacattaaaaacgtccgcaatgtgttattaagttgtctaagcgtcaatttgtttacaccacaatatatcctgccaccagccagccaacagctccccgaccggcagctcggcacaaaatcaccactcgatacaggcagcccatcag SEQ ID NO: 5: Plasmid backbone insertion containing virG gene of pNMD062 ctgtcgatcagatctggctcgcggcggacgcacgacgccggggcgagaccataggcgatctcctaaatcaatagtagctgtaacctcgaagcgtttcacttgtaacaacgattgagaatttttgtcataaaattgaaatacttggttcgcatttttgtcatccgcggtcagccgcaattctgacgaactgcccatttagctggagatgattgtacatccttcacgtgaaaatttctcaagcgctgtgaacaagggttcagattttagattgaaaggtgagccgttgaaacacgttcttcttgtcgatgacgacgtcgctatgcggcatcttattattgaataccttacgatccacgccttcaaagtgaccgcggtagccgacagcacccagttcacaagagtactctcttccgcgacggtcgatgtcgtggttgttgatctaaatttaggtcgtgaagatgggctcgagatcgttcgtaatctggcggcaaagtctgatattccaatcataattatcagtggcgaccgccttgaggagacggataaagttgttgcactcgagctaggagcaagtgattttatcgctaagccgttcagtatcagagagtttctagcacgcattcgggttgccttgcgcgtgcgccccaacgttgtccgctccaaagaccgacggtctttttgttttactgactggacacttaatctcaggcaacgtcgcttgatgtccgaagctggcggtgaggtgaaacttacggcaggtgagttcaatcttctcctcgcgtttttagagaaaccccgcgacgttctatcgcgcgagcaacttctcattgccagtcgagtacgcgacgaggaggtttatgacaggagtatagatgttctcattttgaggctgcgccgcaaacttgaggcggatccgtcaagccctcaactgataaaaacagcaagaggtgccggttatttctttgacgcggacgtgcaggtttcgcacggggggacgatggcagcctaagatcgacag SEQ ID NO: 6: Plasmid backbone insertion containing virG gene of pNMD063, pNMD2190 ctgtcgatcagatctggctcgcggcggacgcacgacgccggggcgagaccataggcgatctcctaaatcaatagtagctgtaacctcgaagcgtttcacttgtaacaacgattgagaatttttgtcataaaattgaaatacttggttcgcatttttgtcatccgcggtcagccgcaattctgacgaactgcccatttagctggagatgattgtacatccttcacgtgaaaatttctcaagtgctgtgaacaagggttcagattttagattgaaaggtgagccgttgaaacacgttcttcttgtcgatgacgacgtcgctatgcggcatcttattattgaataccttacgatccacgccttcaaagtgaccgcggtagccgacagcacccagttcacaagagtactctcttccgcgacggtcgatgtcgtggttgttgatctagatttaggtcgtgaagatgggctcgagatcgttcgtaatctggcggcaaagtctgatattccaatcataattatcagtggcgaccgccttgaggagacggataaagttgttgcactcgagctaggagcaagtgattttatcgctaagccgttcagtatcagagagtttctagcacgcattcgggttgccttgcgcgtgcgccccaacgttgtccgctccaaagaccgacggtctttttgttttactgactggacacttaatctcaggcaacgtcgcttgatgtccgaagctggcggtgaggtgaaacttacggcaggtgagttcaatcttctcctcgcgtttttagagaaaccccgcgacgttctatcgcgcgagcaacttctcattgccagtcgagtacgcgacgaggaggtttatgacaggagtatagatgttctcattttgaggctgcgccgcaaacttgaggcggatccgtcaagccctcaactgataaaaacagcaagaggtgccggttatttctttgacgcggacgtgcaggtttcgcacggggggacgatggcagcctaagatcgacag SEQ ID NO: 7: full-length nucleotide sequence of pNMD1971 ttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcggtgtcatctatgttactagatcgacctgcaggcatgccaattccaatcccacaaaaatctgagcttaacagcacagttgctcctctcagagcagaatcgggtattcaacaccctcatatcaactactacgttgtgtataacggtccacatgccggtatatacgatgactggggttgtacaaaggcggcaacaaacggcgttcccggagttgcacacaagaaatttgccactattacagaggcaagagcagcagctgacgcgtacacaacaagtcagcaaacagacaggttgaacttcatccccaaaggagaagctcaactcaagcccaagagctttgctaaggccctaacaagcccaccaaagcaaaaagcccactggctcacgctaggaaccaaaaggcccagcagtgatccagccccaaaagagatctcctttgccccggagattacaatggacgatttcctctatctttacgatctaggaaggaagttcgaaggtgaaggtgacgacactatgttcaccactgataatgagaaggttagcctcttcaatttcagaaagaatgctgacccacagatggttagagaggcctacgcagcaggtctcatcaagacgatctacccgagtaacaatctccaggagatcaaataccttcccaagaaggttaaagatgcagtcaaaagattcaggactaattgcatcaagaacacagagaaagacatatttctcaagatcagaagtactattccagtatggacgattcaaggcttgcttcataaaccaaggcaagtaatagagattggagtctctaaaaaggtagttcctactgaatctaaggccatgcatggagtctaagattcaaatcgaggatctaacagaactcgccgtgaagactggcgaacagttcatacagagtcttttacgactcaatgacaagaagaaaatcttcgtcaacatggtggagcacgacactctggtctactccaaaaatgtcaaagatacagtctcagaagaccaaagggctattgagacttttcaacaaaggataatttcgggaaacctcctcggattccattgcccagctatctgtcacttcatcgaaaggacagtagaaaaggaaggtggctcctacaaatgccatcattgcgataaaggaaaggctatcattcaagatctctctgccgacagtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgacatctccactgacgtaagggatgacgcacaatcccactatccttcgcaagacccttcctctatataaggaagttcatttcatttggagaggacacgctcgagtataagagctctatttttacaacaattaccaacaacaacaaacaacaaacaacattacaattacatttacaattaccatggaacgagctatacaaggaaacgatgctagggaacaagcttatggtgaacgttggaatggaggatcaggaagttccacttctcccttcaaacttcctgacgaaagtccgagttggactgagtggcggctacataacgatgagacgatttcgaatcaagataatccccttggtttcaaggaaagctggggtttcgggaaagttgtatttaagagatatctcagatacgacgggacggaaacttcactgcacagagtccttggatcttggacgggagattcggttaactatgcagcatctcgatttctcggtttcgaccagatcggatgtacctatagtattcggtttcgaggagttagtgtcaccatttctggagggtcgcgaactcttcagcatctcagtgaaatggcaattcggtctaagcaagaactgctacagcttaccccagtcaaagtggaaagtgatgtatcaagaggatgccctgaaggtgttgaaaccttcgaagaagaaagcgagtaaggatcctctagagtcctgctttaatgagatatgcgagacgcctatgatcgcatgatatttgctttcaattctgttgtgcacgttgtaaaaaacctgagcatgtgtagctcagatccttaccgccggtttcggttcattctaatgaatatatcacccgttactatcgtatttttatgaataatattctccgttcaatttactgattgtaccctactacttatatgtacaatattaaaatgaaaacaatatattgtgctgaataggtttatagcgacatctatgatagagcgccacaataacaaacaattgcgttttattattacaaatccaattttaaaaaaagcggcagaaccggtcaaacctaaaagactgattacataaatcttattcaaatttcaaaagtgccccaggggctagtatctacgacacaccgagcggcgaactaataacgctcactgaagggaactccggttccccgccggcgcgcatgggtgagattccttgaagttgagtattggccgtccgctctaccgaaagttacgggcaccattcaacccggtccagcacggcggccgggtaaccgacttgctgccccgagaattatgcagcatttttttggtgtatgtgggccccaaatgaagtgcaggtcaaaccttgacagtgacgacaaatcgttgggcgggtccagggcgaattttgcgacaacatgtcgaggctcagcaggacctgcataagctcttctgtcagcgggcccactgcatccaccccagtacattaaaaacgtccgcaatgtgttattaagttgtctaagcgtcaatttgtttacaccacaatatatcctgccaccagccagccaacagctccccgaccggcagctcggcacaaaatcaccactcgatacaggcagcccatcagtcagatcaggatctcctttgcgacgctcaccgggctggttgccctcgccgctgggctggcggccgtctatggccctgcaaacgcgccagaaacgccgtcgaagccgtgtgcgagacaccgcggccgccggcgttgtggatacctcgcggaaaacttggccctcactgacagatgaggggcggacgttgacacttgaggggccgactcacccggcgcggcgttgacagatgaggggcaggctcgatttcggccggcgacgtggagctggccagcctcgcaaatcggcgaaaacgcctgattttacgcgagtttcccacagatgatgtggacaagcctggggataagtgccctgcggtattgacacttgaggggcgcgactactgacagatgaggggcgcgatccttgacacttgaggggcagagtgctgacagatgaggggcgcacctattgacatttgaggggctgtccacaggcagaaaatccagcatttgcaagggtttccgcccgtttttcggccaccgctaacctgtcttttaacctgcttttaaaccaatatttataaaccttgtttttaaccagggctgcgccctgtgcgcgtgaccgcgcacgccgaaggggggtgcccccccttctcgaaccctcccggcccgctaacgcgggcctcccatccccccaggggctgcgcccctcggccgcgaacggcctcaccccaaaaatggcagcgctggccaattcgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatggctaaaatgagaatatcaccggaattgaaaaaactgatcgaaaaataccgctgcgtaaaagatacggaaggaatgtctcctgctaaggtatataagctggtgggagaaaatgaaaacctatatttaaaaatgacggacagccggtataaagggaccacctatgatgtggaacgggaaaaggacatgatgctatggctggaaggaaagctgcctgttccaaaggtcctgcactttgaacggcatgatggctggagcaatctgctcatgagtgaggccgatggcgtcctttgctcggaaggtatgaagatgaacaaagccctgaaaagattatcgagctgtatgcggagtgcatcaggctctttcactccatcgacatatcggattgtccctatacgaatagcttagacagccgcttagccgaattggattacttactgaataacgatctggccgatgtggattgcgaaaactgggaagaagacactccatttaaagatccgcgcgagctgtatgattttttaaagacggaaaagcccgaagaggaacttgtcttttcccacggcgacctgggagacagcaacatctttgtgaaagatggcaaagtaagtggctttattgatcttgggagaagcggcagggcggacaagtggtatgacattgccttctgcgtccggtcgatcagggaggatatcggggaagaacagtatgtcgagctattttttgacttactggggatcaagcctgattgggagaaaataaaatattatattttactggatgaattgttttagctgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggcagatcctagatgtggcgcaacgatgccggcgacaagcaggagcgcaccgacttcttccgcatcaagtgttttggctctcaggccgaggcccacggcaagtatttgggcaaggggtcgctggtattcgtgcagggcaagattcggaataccaagtacgagaaggacggccagacggtctacgggaccgacttcattgccgataaggtggattatctggacaccaaggcaccaggcgggtcaaatcaggaataagggcacattgccccggcgtgagtcggggcaatcccgcaaggagggtgaatgaatcggacgtttgaccggaaggcatacaggcaagaactgatcgacgcggggttttccgccgaggatgccgaaaccatcgcaagccgcaccgtcatgcgtgcgccccgcgaaaccttccagtccgtcggctcgatggtccagcaagctacggccaagatcgagcgcgacagcgtgcaactggctccccctgccctgcccgcgccatcggccgccgtggagcgttcgcgtcgtctcgaacaggaggcggcaggtttggcgaagtcgatgaccatcgacacgcgaggaactatgacgaccaagaagcgaaaaaccgccggcgaggacctggcaaaacaggtcagcgaggccaagcaggccgcgttgctgaaacacacgaagcagcagatcaaggaaatgcagctttccttgttcgatattgcgccgtggccggacacgatgcgagcgatgccaaacgacacggcccgctctgcccgctctgccctgttcaccacgcgcaacaagaaaatcccgcgcgaggcgctgcaaaacaaggtcattttccacgtcaacaaggacgtgaagatcacctacaccggcgtcgagctgcgggccgacgatgacgaactggtgtggcagcaggtgttggagtacgcgaagcgcacccctatcggcgagccgatcaccttcacgttctacgagctttgccaggacctgggctggtcgatcaatggccggtattacacgaaggccgaggaatgcctgtcgcgcctacaggcgacggcgatgggcttcacgtccgaccgcgttgggcacctggaatcggtgtcgctgctgcaccgcttccgcgtcctggaccgtggcaagaaaacgtcccgttgccaggtcctgatcgacgaggaaatcgtcgtgctgtttgctggcgaccactacacgaaattcatatgggagaagtaccgcaagctgtcgccgacggcccgacggatgttcgactatttcagctcgcaccgggagccgtacccgctcaagctggaaaccttccgcctcatgtgcggatcggattccacccgcgtgaagaagtggcgcgagcaggtcggcgaagcctgcgaagagttgcgaggcagcggcctggtggaacacgcctgggtcaatgatgacctggtgcattgcaaacgctagggccttgtggggtcagttccggctgggggttcagcagccagcgcctgatctggggaaccctgtggttggcacatacaaatggacgaacggataaaccttttcacgcccttttaaatatccgattattctaataaacgctcttttctcttaggtttacccgccaatatatcctgtcaaacactgatagtttaaactgaaggcgggaaacgacaatctgatctaagctaggcatggaattccaatcccacaaaaatctgagcttaacagcacagttgctcctctcagagcagaatcgggtattcaacaccctcatatcaactactacgttgtgtataacggtccacatgccggtatatacgatgactggggttgtacaaaggcggcaacaaacggcgttcccggagttgcacacaagaaatttgccactattacagaggcaagagcagcagctgacgcgtacacaacaagtcagcaaacagacaggttgaacttcatccccaaaggagaagctcaactcaagcccaagagctttgctaaggccctaacaagcccaccaaagcaaaaagcccactggctcacgctaggaaccaaaaggcccagcagtgatccagccccaaaagagatctcctttgccccggagattacaatggacgatttcctctatctttacgatctaggaaggaagttcgaaggtgaaggtgacgacactatgttcaccactgataatgagaaggttagcctcttcaatttcagaaagaatgctgacccacagatggttagagaggcctacgcagcaggtctcatcaagacgatctacccgagtaacaatctccaggagatcaaataccttcccaagaaggttaaagatgcagtcaaaagattcaggactaattgcatcaagaacacagagaaagacatatttctcaagatcagaagtactattccagtatggacgattcaaggcttgcttcataaaccaaggcaagtaatagagattggagtctctaaaaaggtagttcctactgaatctaaggccatgcatggagtctaagattcaaatcgaggatctaacagaactcgccgtgaagactggcgaacagttcatacagagtcttttacgactcaatgacaagaagaaaatcttcgtcaacatggtggagcacgacactctggtctactccaaaaatgtcaaagatacagtctcagaagaccaaagggctattgagacttttcaacaaaggataatttcgggaaacctcctcggattccattgcccagctatctgtcacttcatcgaaaggacagtagaaaaggaaggtggctcctacaaatgccatcattgcgataaaggaaaggctatcattcaagatctctctgccgacagtggtcccaaagatggacccccacccacgaggagcatcgtggaaaaagaagacgttccaaccacgtcttcaaagcaagtggattgatgtgacatctccactgacgtaagggatgacgcacaatcccactatccttcgcaagacccttcctctatataaggaagttcatttcatttggagaggacacgctcgagtataagagctcatttttacaacaattaccaacaacaacaaacaacaaacaacattacaattacatttacaattatcgatgggtcagtcccttatgttacgtcctgtagaaaccccaacccgtgaaatcaaaaaactcgacggcctgtgggcattcagtctggatcgcgaaaactgtggaattgatcagcgttggtgggaaagcgcgttacaagaaagccgggcaattgctgtgccaggcagttttaacgatcagttcgccgatgcagatattcgtaattatgcgggcaacgtctggtatcagcgcgaagtctttataccgaaaggtaagtagtgtttttggataactgagtttgcctatgattttgtatttactgagatgtttgtcctctttgtgcaggttgggcaggccagcgtatcgtgctgcgtttcgatgcggtcactcattacggcaaagtgtgggtcaataatcaggaagtgatggagcatcagggcggctatacgccatttgaagccgatgtcacgccgtatgttattgccgggaaaagtgtacgtatcaccgtttgtgtgaacaacgaactgaactggcagactatcccgccgggaatggtgattaccgacgaaaacggcaagaaaaagcagtcttacttccatgatttctttaactatgccggaatccatcgcagcgtaatgctctacaccacgccgaacacctgggtggacgatatcaccgtggtgacgcatgtcgcgcaagactgtaaccacgcgtctgttgactggcaggtggtggccaatggtgatgtcagcgttgaactgcgtgatgcggatcaacaggtggttgcaactggacaaggcactagcgggactttgcaagtggtgaatccgcacctctggcaaccgggtgaaggttatctctatgaactgtgcgtcacagccaaaagccagacagagtgtgatatctacccgcttcgcgtcggcatccggtcagtggcagtgaagggccaacagttcctgattaaccacaaaccgttctactttactggctttggtcgtcatgaagatgcggacttacgtggcaaaggattcgataacgtgctgatggtgcacgaccacgcattaatggactggattggggccaactcctaccgtacctcgcattacccttacgctgaagagatgctcgactgggcagatgaacatggcatcgtggtgattgatgaaactgctgctgtcggctttaacctctctttaggcattggtttcgaagcgggcaacaagccgaaagaactgtacagcgaagaggcagtcaacggggaaactcagcaagcgcacttacaggcgattaaagagctgatagcgcgtgacaaaaaccacccaagcgtggtgatgtggagtattgccaacgaaccggatacccgtccgcaaggtgcacgggaatatttcgcgccactggcggaagcaacgcgtaaactcgacccgacgcgtccgatcacctgcgtcaatgtaatgttctgcgacgctcacaccgataccatcagcgatctctttgatgtgctgtgcctgaaccgttattacggatggtatgtccaaagcggcgatttggaaacggcagagaaggtactggaaaaagaacttctggcctggcaggagaaactgcatcagccgattatcatcaccgaatacggcgtggatacgttagccgggctgcactcaatgtacaccgacatgtggagtgaagagtatcagtgtgcatggctggatatgtatcaccgcgtctttgatcgcgtcagcgccgtcgtcggtgaacaggtatggaatttcgccgattttgcgacctcgcaaggcatattgcgcgttggcggtaacaagaaagggatcttcactcgcgaccgcaaaccgaagtcggcggcttttctgctgcaaaaacgctggactggcatgaacttcggtgaaaaaccgcagcagggaggcaaacaatgaatcaacaactctcctggcgcaccatcgtcggctacagcctcgggaattgggatcctctagagtcaagcagatcgttcaaacatttggcaataaagtttc

The invention claimed is:
 1. A process of transiently transfecting aNicotiana plant or leaves on said plant, comprising contacting saidplant or said leaves with a suspension comprising Agrobacterium cells ofstrain CryX having accession no. DSM25686, wherein the transienttransfection efficiency obtainable with strain CryX is higher than withstrain KYRT1 when infiltrating Nicotiana benthamiana leaves usingneedleless syringe with dilutions of agrobacterial cultures of bothstrains harboring a green fluorescent protein (GFP) expression tobaccomosaic virus (TMV)-based vector and comparing the intensity of GFPfluorescence.
 2. A process of transiently expressing a DNA sequence ofinterest in a Nicotiana plant, comprising contacting said plant orleaves on said plant with a suspension comprising Agrobacterium cells ofstrain CryX having accession no. DSM25686, wherein the transienttransfection efficiency obtainable with strain CryX is higher than withstrain KYRT1 when infiltrating Nicotiana benthamiana leaves usingneedleless syringe with dilutions of agrobacterial cultures of bothstrains harboring a GFP expression TMV-based vector and comparing theintensity of GFP fluorescence.
 3. The process according to claim 1,wherein said strain CryX contains a binary vector having T-DNAcomprising a DNA sequence of interest to be transfected into cells ofsaid plant or leaves.
 4. The process according to claim 3, wherein saidbinary vector comprises a virG gene expressible in said strain CryX. 5.The process according to claim 4, wherein said virG gene encodes a VirGprotein from Agrobacterium tumefaciens strain LBA4404 of SEQ ID NO: 1,or is an N54D mutant of the VirG protein encoded by the virG gene fromA. tumefaciens strain LBA4404.
 6. The process according to claim 1,wherein said Nicotiana plant or leaves on said plant are contacted withsaid suspension by spraying or by vacuum infiltrating said plant orleaves on said plant with said suspension.
 7. The process according toclaim 1, wherein said Nicotiana plant is a Nicotiana benthamiana.